Methods for Treating Osteogenesis Imperfecta

ABSTRACT

The present invention provides methods for treating and improving the symptoms of osteogenesis imperfecta (OI) in a subject by administering to the subject a therapeutically effective amount of a binding agent that binds to transforming growth factor beta (TGFβ).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/459,023, filed Jul. 1, 2019, which is a continuation of U.S. patentapplication Ser. No. 15/427,920, filed Feb. 8, 2017, now U.S. Pat. No.10,377,819, issued Aug. 13, 2019, which is a continuation of U.S. patentapplication Ser. No. 14/772,708, filed Sep. 3, 2015, now U.S. Pat. No.9,598,486, issued Mar. 21, 2017, which is a 35 U.S.C. § 371 NationalStage filing of International Patent Application PCT/US2014/031279,filed Mar. 20, 2014, which claims priority to U.S. Provisional PatentApplication 61/803,647, filed Mar. 20, 2013, U.S. Provisional PatentApplication 61/875,399, filed Sep. 9, 2013, and U.S. Provisional PatentApplication 61/883,151, filed Sep. 26, 2013. The contents of each of theaforementioned priority applications are incorporated herein byreference in their entirety.

GOVERNMENT FUNDING

This invention was made with government support under P01 HD070394, P01HD022657 & R01 DE017713 awarded by the National Institutes of Health.The government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing that has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. The ASCII copy, created on Dec. 8, 2021, isnamed 022548_C3018_SL.txt and is 36,652 bytes in size.

FIELD OF THE INVENTION

The present invention relates to methods for treating osteogenesisimperfecta (OI). More specifically, the invention relates to methods fortreating OI using a binding protein, e.g., an antibody orantigen-binding fragment thereof, which specifically binds to humantransforming growth factor beta (TGFβ) or isoforms thereof.

BACKGROUND OF THE INVENTION

Osteogenesis imperfecta (OI), also known as “brittle bone disease” orLobstein syndrome, is a debilitating and rare congenital bone diseasethat affects about one in every 15,000 people. Though phenotypes varyamong OI types, common symptoms include incomplete ossification of bonesand teeth, reduced bone mass, brittle bones, and pathologic fractures.These common symptoms of OI are thought to be caused by gene mutationswhich result in deficiencies in Type-I collagen or other proteinsinvolved in bone matrix deposition or homeostasis. As a result of thesesymptoms and the propensity for fatal bone fractures and complications,life expectancy of OI patients is reduced as compared to the generalpopulation. Accordingly, there clearly exists an urgent need in the artto develop effective treatments for OI.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A (Western blot) and 1B-1F (graphs) demonstrate that TGFβsignaling is elevated in bones from Crtap^(−/−) mice, as compared towild type controls.

FIGS. 2A (luminescent picture) and 2B-C(graphs) show that Crtap^(−/−)mice crossed with TGFβ reporter mice exhibit higher TGFβ activitycompared to wild type controls.

FIG. 3 is a μCT image of vertebrae from Crtap^(−/−) mice treated withthe mouse pan-specific anti-TGFβ antibody 1D11.

FIG. 4 is a graph that contains quantitative measurements of vertebraefrom Crtap^(−/−) mice treated with the mouse pan-specific anti-TGFβantibody 1D11.

FIGS. 5A-B are a histomorphometric analysis of vertebrae fromCrtap^(−/−) mice treated with the mouse pan-specific anti-TGFβ antibody1D11.

FIGS. 6A and 6B are graphs that demonstrates the biomechanicalproperties (FIG. 6A—maximum load, and FIG. 6B—stiffness) of femurs fromCrtap^(−/−) mice treated with the mouse pan-specific anti-TGFβ antibody1D11, as determined by a three-point bending test.

FIGS. 7A and 7B (micrograph images) and 7C (graph) demonstrate the lungphenotype of Crtap^(−/−) mice treated with the mouse pan-specificanti-TGFβ antibody 1D11.

FIG. 8 are micrograph images of Crtap^(−/−) and WT lungs stained with ananti-decorin antibody.

FIG. 9 is a graph depicting a decorin binding assay.

FIGS. 10A-F are a group of graphs, photos, and images that showincreased TGFβ signaling in Crtap^(−/−) mice. FIG. 10A is a series ofthree graphs that show the results of quantitative RT-PCR of TGFβ targetgenes p21, PAI-1, and Col1a1. The graphs indicate increased TGFβsignaling in calvarial bone of P3 WT and Crtap^(−/−) mice. Results areshown as fold change of the mean of WT group±SD; n=5 per group, *p<0.05.FIG. 10B is a photograph of a Western blot analysis of P3 calvarialprotein extracts, which shows increased amounts of activated Smad2(pSmad2) relative to total Smad2 protein in Crtap^(−/−) versus WT mice,suggesting increased TGFβ-signaling; n=3 per group. FIG. 10C is a graphshowing the quantification of the Western blot shown in FIG. 10B.Results are shown as fold change of the mean of WT group±SD, *p<0.05.FIG. 10D is an image showing increased bioluminescence in regions thatoverlap with skeletal structures in Crtap^(−/−) compared with WT micethat were intercrossed to TGFβ-reporter mice (SBE-Luc mice).Representative image of 3 litters at P10 is shown. In 3 littersCrtap^(−/−) mice show a mean 2.86 fold (SD±0.34) bioluminescence signalat the head/calvaria compared with WT mice (scale bar=1 cm). FIG. 10E isa graph that shows, using a TGFβ reporter cell line, TGFβ activity wasassessed in conditioned medium from WT and Crtap^(−/−) bone marrowstromal cells cultured under osteogenic conditions for 3 days,demonstrating greater TGFβ activity compared with the medium from WTcells. Results are shown as fold change of the mean of WT group±SD, n=5per group, *p<0.05. FIG. 10F is two pictures showing immunostaining oflungs (P10) for pSmad2, which shows increased intracellular staining inWT and Crtap^(−/−) mice (40× magnification). Representative images ofn=3 mice per group are shown (scale bar=20 μm).

FIGS. 11A-D are a group of photos and graphs showing phenotypiccorrection of Crtap^(−/−) mice after treatment with the TGFβneutralizing antibody 1D11. FIG. 11A is a series of three MicroCT imagesof L4 vertebral bodies of 16-week-old wildtype (WT), controlantibody-treated Crtap^(−/−), and 1D11-treated Crtap^(−/−) mice aftertreatment for 8 weeks. FIG. 11B is a group of three graphs showing theresults of MicroCT of L4 vertebral bodies, which demonstrates increasedbone volume/total volume (BV/TV), trabecular number (Tb.N), andthickness (Tb.Th) in WT, control Crtap^(−/−) and 1D11 treatedCrtap^(−/−) mice. Results are shown as means±SDs, n=8 per group, *p<0.05for Crtap^(−/−) 1 D11 vs. Crtap^(−/−) control, +p<0.05 for Crtap^(−/−)vs. WT. FIG. 11C is a group of three graphs showing the results ofhistomorphometric analysis of L4 vertebrae, which shows increasedosteoclast (N.Oc/BS) and osteoblast (N.Ob/BS) numbers per bone surfacein Crtap^(−/−) mice compared with WT. Reduced osteoblast and osteoclastnumbers after treatment with 1D11 indicates effective suppression ofaccelerated bone remodeling in Crtap^(−/−) mice. Increased numbers ofosteocytes per bone area (N.Ot/B.Ar) in Crtap^(−/−) mice are reduced toWT level after 1D11 treatment. Results are shown as means±SDs, n=6 pergroup, *p<0.05 for Crtap^(−/−) 1 D11 vs. Crtap^(−/−) control, +p<0.05for Crtap^(−/−) vs. WT. FIG. 11D is a series of three pictures showinghematoxylin/eosin staining of inflated lungs of 16-week-old wildtype(WT), control Crtap^(−/−), and 1D11-treated Crtap^(−/−) mice aftertreatment for 8 weeks. Crtap^(−/−) control mice show an increase indistal airway space compared with WT mice. After treatment with 1D11,there is a reduction of the distal airway diameter compared with controlCrtap^(−/−) mice. Representative images of n=8 mice per group are shown(scale bar=100 μm). FIG. 11E is a graph showing quantification of thedistance between alveolar structures by the mean-linear-intercept (MLI)method, which demonstrates a significant reduction of the distal airwayspace in 1D11-treated Crtap^(−/−) mice compared with controlantibody-treated Crtap^(−/−) and WT mice. Results are shown asmeans±SDs, n=8 mice per group, 10 images analyzed per mouse, *p<0.05 forCrtap^(−/−) 1D11 vs. Crtap^(−/−) control, +p<0.05 for Crtap^(−/−) vs.WT.

FIGS. 12A-B are a series of graphs that show that decorin binding totype I collagen overlaps the P986 3Hyp site and is reduced in type Icollagen of Crtap^(−/−) mice. FIG. 12A is a group of three graphs thatshow the results of quantitative RT-PCR of calvarial bone of P3 mice,which shows no difference in RNA expression of the small leucine-richproteoglycans decorin (Dcn), biglycan (Bgn), and asporin (Aspn) incalvarial bone of Crtap^(−/−) mice compared with WT. Results are givenas fold change of the mean of WT group±SD, n=5 per group. FIG. 12B is agraph that shows the results of surface plasmon resonance analysis,which indicates that binding of recombinant decorin core protein to typeI collagen of Crtap^(−/−) mice is approximately 45% less compared withWT type I collagen. Three independent experiments using 3, 5, and 12 μMof decorin were performed. Response units (RU) of total amount decorinbound normalized to type I collagen immobilized on the chip are shown.Mean reduction of decorin binding to Crtap^(−/−) type I collagen is44.6±7.9%.

FIGS. 13A-E are a series of graphs and photos showing inhibition ofincreased TGFβ signaling improves the bone phenotype in a mouse model ofdominant OI resulting from a G610C mutation in the Col1a2 gene(Col1a2^(tm1.1Mcbr)). FIG. 13A is two graphs showing the results ofquantitative RT-PCR of TGFβ target genes p21 and PAI-1, which indicatesincreased TGFβ signaling in calvarial bone of P3 WT andCol1a2^(tm1.1Mcbr) mice. Results are shown as fold change of the mean ofWT group±SD; n=3 per group, *p<0.05. FIG. 13B is a photo of the resultsof Western blot analysis, which shows increased levels of activatedSmad2 (pSmad2) relative to total levels of Smad2 protein in P3 calvariaof WT and Col1a2^(tm1.1Mcbr) mice compared with WT, suggesting increasedTGFβ-signaling; n=3 per group. FIG. 13C is a graph showing thequantification of the Western blot seen in FIG. 13B. Results are shownas fold change of the mean of WT group±SD; *p<0.05. FIG. 13D are aseries of photos of MicroCT images of L4 vertebral bodies of 16-week-oldwildtype (WT), control antibody-treated Col1a2^(tm1.1Mcbr) and1D11-treated Col1a2^(tm1.1Mcbr) mice after treatment for 8 weeks. FIG.13E is a series of graphs of data from MicroCT of L4 vertebral bodies,which shows increased bone volume/total volume (BV/TV), trabecularnumber (Tb.N) and thickness (Tb.Th) in Col1a2^(tm1.1Mcbr) mice aftertreatment with 1D11. Results are shown as means±SDs, n=6 per group,*p<0.05 for Col1a2^(tm1.1Mcbr) 1D11 vs. Col1a2^(tm1.1Mcbr) control,+p<0.05 for Col1a2^(tm1.1Mcbr) vs. WT.

FIG. 14 is a weight curve graph, which shows a reduced weight ofCrtap^(−/−) mice compared with WTs during the study period (p<0.05 forall time points, means±SEs are shown). No statistically significantdifference in weight was observed in 1D11-treated Crtap^(−/−) micecompared to control Crtap^(−/−) mice.

FIGS. 15A-C are a series of graphs and tables showing no effect of TGFβinhibition on the abnormal type I collagen post-translationalmodification in Crtap^(−/−) mice. FIG. 15A is a series of three graphsshowing tandem mass spectra of extracted type I collagen from tibia ofWT, control Crtap^(−/−), and 1D11-treated Crtap^(−/−) mice (16 week oldmice, after treatment for 8 weeks). The sequence in the top graph is SEQID NO: 19, the sequence in the middle graph is SEQ ID NO: 20, and thesequence in the bottom graph is SEQ ID NO: 21. FIG. 15B is a tableshowing a summary of tandem mass spectra analyses. 1D11 treatment didnot significantly affect 3-hydroxylation status of collagen residuePro986 alpha 1(I) in bone samples. Mean of percentage of 3-hydroxylatedresidues (±SD) is shown, n=5 per group. FIG. 15C is a group of threegraphs showing that bone type I collagen of control Crtap^(−/−) and1D11-treated Crtap^(−/−) mice exhibit changes in hydroxylysylpyridinoline (HP) and lysyl pyridinoline crosslinks (LP) levels and anincreased HP/LP ratio compared with WT mice. 1D11 treatment ofCrtap^(−/−) mice did not significantly affect these parameters comparedto control Crtap^(−/−) mice. Results are given as means±SDs, n=4 miceper group, +p<0.05 for Crtap^(−/−) vs. WT.

FIGS. 16A-B are a series of graphs showing serum bone-turnover markersosteocalcin (OCN) and C-terminal cross-linked telopeptide of bonecollagen (CTX) at start (FIG. 16A=8 weeks of age) and end of thetreatment study (FIG. 16B=16 weeks of age). FIG. 16A is two graphsshowing increased OCN and CTX serum levels in 8 week old Crtap^(−/−)compared with WT mice at the start of the study indicate increased boneturnover in Crtap^(−/−) mice. Results are given as means±SDs, n=8 forWT, n=14 for Crtap^(−/−) mice, +p<0.05 for Crtap^(−/−) vs. WT. FIG. 16Bis two graphs that show that at 16 weeks of age 1D11-treated Crtap^(−/−)mice show a trend to reduced serum OCN and significantly reduced CTXserum levels compared with control Crtap^(−/−) mice, indicating asuppression of increased bone turnover by inhibition of TGFβ. Resultsare given as means±SDs, n=8 for WT, n=7 per Crtap^(−/−) group; *p<0.05for Crtap^(−/−) 1 D11 vs. Crtap^(−/−) control, +p<0.05 for Crtap^(−/−)vs. WT.

FIG. 17 is a table showing the results of MicroCT analyses of vertebralbody L4 of WT, control Crtap^(−/−), and 1D11 treated Crtap^(−/−) mice(16 week old mice, after treatment for 8 weeks). Means±SDs are shown forbone volume/tissue volume (BV/TV), trabecular number (Tb.N), trabecularthickness (Tb.Th), trabecular separation (Tb.Sp), and bone mineraldensity of bone volume (BMD BV); n=8 per group, +indicatesKruskal-Wallis one-way ANOVA on ranks where the equal variance testfailed. n.s.=not statistically significant.

FIG. 18 is a table showing the results of MicroCT analyses of trabecularbone in proximal femurs for WT, control Crtap^(−/−), and 1D11-treatedCrtap^(−/−) mice (16 week old mice, after treatment for 8 weeks).Means±SDs are shown for bone volume/tissue volume (BV/TV), trabecularnumber (Tb.N), trabecular thickness (Tb.Th), trabecular separation(Tb.Sp), and bone mineral density of bone volume (BMD BV); n=8 pergroup. +indicates Kruskal-Wallis one-way ANOVA on ranks where the equalvariance test failed. n.s.=not statistically significant.

FIG. 19 is a table showing the results of MicroCT analysis of corticalbone at the femur midshaft for WT, control Crtap^(−/−), and 1D11-treatedCrtap^(−/−) mice (16 week old mice, after treatment for 8 weeks).Means±SDs are shown for cortical thickness, bone mineral density of bonevolume (BMD BV), anterior-posterior (a.p.) diameter, cross-sectionalarea (CSA), and cross-sectional moments of inertia (CSMI) formedio-lateral (m.1.) and anterior-posterior (a.p.) axis; n=8 per group.n.s.=not statistically significant.

FIG. 20 is a table showing the results of biomechanical testing offemurs by 3-point bending (16 week old mice, after treatment for 8weeks). Compared with WT mice, control Crtap^(−/−) mice exhibitsignificantly reduced biomechanical parameters except elastic modulusand elastic displacement. Anti TGFβ-treatment with 1D11 resulted insignificant improvements of maximum load and ultimate strength inCrtap^(−/−) femurs, indicating increased whole bone and tissue strength.However, no significant changes in post-yield displacement wereobserved, indicating that 1D11 did not affect the increased brittlenessof the OI bone. N=6 for WT, n=4 for control Crtap^(−/−) and n=3 for 1D11treated Crtap^(−/−) mice. n.s.=not statistically significant.

FIG. 21 is a table showing the results of histomorphometry analyses ofL4 vertebral bodies of WT, control Crtap^(−/−), and 1D11-treatedCrtap^(−/−) mice (16 week old mice, after treatment for 8 weeks).Means±SDs are shown for bone volume/tissue volume (BV/TV), trabecularnumber (Tb.N), trabecular thickness (Tb.Th), trabecular separation(Tb.Sp), number of osteoclasts/bone surface (N.Oc/BS), osteoclastsurface/bone surface (Oc.S/BS), number of osteoblasts/bone surface(N.Ob/BS), osteoblast surface/bone surface (Oc.S/BS), and number ofosteocytes/bone area (N.Ot/B.Ar); n=6 per group. +indicatesKruskal-Wallis one way ANOVA on ranks where equal variance test failed.n.s.=not statistically significant.

FIG. 22 is a table showing the results of MicroCT analyses of vertebralbody L4 of WT, control Col1a2^(tm1.1Mcbr) and 1D11 treatedCol1a2^(tm1.1Mcbr) mice (16 week old mice, after treatment for 8 weeks).Means±SDs are shown for bone volume/tissue volume (BV/TV), trabecularnumber (Tb.N), trabecular thickness (Tb.Th), trabecular separation(Tb.Sp), and bone mineral density of bone volume (BMD BV); n=6 pergroup, n.s.=not statistically significant.

FIG. 23 is a graph showing surface plasmon resonance analysis measuringthe binding of recombinant decorin core protein to type I collagen of WTand Crtap^(−/−) mice. Three technical replicates at each of theindicated concentrations of decorin were performed from two independentbiological replicates (♦ replicate 1, ▴ replicate 2). Results are shownas the percentage of the mean of WT (bars indicate mean per group).

FIG. 24 (micrograph images) demonstrate immunostaining for decorin inthe distal femur metaphysis of WT and Crtap^(−/−) mice at 20λ, n=3 pergenotype, scale bars=100 μm (Panels A-C) and 40× magnification, n=3 pergenotype, scale bars=50 μm (Panels D-F). Control femurs were incubatedin secondary antibody only.

FIG. 25 (micrograph images) demonstrate immunostaining for TGFβ1 in thedistal femur metaphysis of WT and Crtap^(−/−) mice at 20λ, n=3 pergenotype, scale bars=100 μm (Panels A-C) and 40× magnification, n=3 pergenotype, scale bars=50 μm (Panels D-F). Control femurs were incubatedin secondary antibody only.

FIG. 26 (micrograph images) demonstrate immunostaining for TGFβ1 in thedistal femur metaphysis of WT and Col1a2^(tm1.1Mcbr) mice at 20λ, n=3per genotype, scale bars=100 μm (Panels A-C) and 40× magnification, n=3per genotype, scale bars=50 μm (Panels D-F). Control femurs wereincubated in secondary antibody only.

SUMMARY OF THE INVENTION

The present invention relates to methods for effectively treatingosteogenesis imperfecta (OI). More specifically, the invention relatesto methods for treating OI using a binding protein such as antibody oran antigen-binding fragment thereof that specifically binds totransforming growth factor beta (TGFβ) or an isoform thereof.Preferably, the binding protein is “pan-specific” and binds to all threehuman isoforms of TGFβ, i.e., TGFβ1, TGFβ2, and TGFβ3. More preferably,the binding protein specifically binds to and neutralizes human TGFβ1,TGFβ2, and TGFβ3. In one aspect, the invention provides a method fortreating OI in a subject in need thereof comprising administering to thesubject a therapeutically effective amount of an antibody or anantigen-binding fragment thereof that specifically binds to TGFβ.

In one embodiment, the antibody or antigen-binding fragment thereofcomprises a heavy chain variable region comprising three complementaritydetermining regions (CDRs) having amino acid sequences selected from thegroup consisting of SEQ ID NOs: 4, 5, and 6; and a light chain variableregion comprising three CDRs having amino acid sequences selected fromthe group consisting of SEQ ID NOs: 7, 8, and 9.

In another embodiment, the antibody or antigen-binding fragment thereofcomprises a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 10, and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 11.

In one embodiment, the antibody or antigen-binding fragment thereoffurther comprises a human IgG4 constant region. In one embodiment, thehuman IgG4 constant region comprises the amino acid sequence of SEQ IDNO: 12. In another embodiment, the antibody or antigen-binding fragmentthereof further comprises a human κ light chain constant region. Inanother embodiment, the human κ light chain constant region comprisesthe amino acid sequence of SEQ ID NO: 13. In another embodiment, theantibody or antigen-binding fragment thereof further comprises a humanIgG4 constant region, and a human κ light chain constant region.

In another embodiment, the human IgG4 constant region comprises theamino acid sequence of SEQ ID NO: 12, and the human κ light chainconstant region comprises the amino acid sequence of SEQ ID NO: 13. Inanother embodiment, the antibody comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 14. In another embodiment, theantibody comprises a light chain comprising the amino acid sequence ofSEQ ID NO: 15. In another embodiment, the antibody comprises a heavychain comprising the amino acid sequence of SEQ ID NO: 14, and a lightchain comprising the amino acid sequence of SEQ ID NO: 15.

In another embodiment, the antibody or antigen-binding fragment thereofbinds to human TGFβ1, TGFβ2, and TGFβ3. In another embodiment, theantibody or antigen-binding fragment thereof neutralizes human TGFβ1,TGFβ2, and TGFβ3.

In another embodiment, the antibody or antigen-binding fragment thereofimproves a bone parameter selected from the group consisting of bonevolume density (BV/TV), total bone surface (BS), bone surface density(BS/BV), trabecular number (Tb.N), trabecular thickness (Tb.Th),trabecular spacing (Tb. Sp), and total volume (Dens TV).

In another embodiment, the antibody or antigen-binding fragment thereofinhibits bone resorption.

In another embodiment, the antibody or antigen-binding fragment thereofreduces a serum biomarker of bone resorption selected from the groupconsisting of urinary hydroxyproline, urinary total pyridinoline (PYD),urinary free deoxypyridinoline (DPD), urinary collagen type-Icross-linked N-telopeptide (NTX), urinary or serum collagen type-Icross-linked C-telopeptide (CTX), bone sialoprotein (BSP), osteopontin(OPN), and tartrate-resistant acid phosphatase 5b (TRAP).

In another embodiment, the antibody or antigen-binding fragment thereofincreases a serum biomarker of bone deposition selected from the groupconsisting of as total alkaline phosphatase, bone-specific alkalinephosphatase, osteocalcin, and type-I procollagen(C-terminal/N-terminal).

In another embodiment, the antibody or antigen-binding fragment thereofinhibits bone resorption. In another embodiment, the antibody orantigen-binding fragment thereof promotes bone deposition. In anotherembodiment, the antibody or antigen-binding fragment thereof improvesthe function of a non-skeletal organ affected by OI selected from thegroup consisting of hearing function, lung function, and kidneyfunction.

In another aspect, the invention provides a method for treating OI in asubject in need thereof comprising administering to the subject atherapeutically effective amount of an antibody or an antigen-bindingfragment thereof that binds to TGFβ, wherein the antibody comprises aheavy chain comprising the amino acid sequence of SEQ ID NO: 14, and alight chain comprising the amino acid sequence of SEQ ID NO: 15.

In another aspect, the invention provides a method for treating OI in asubject in need thereof comprising administering to the subject atherapeutically effective amount of an antibody or an antigen-bindingfragment thereof that binds to TGFβ in combination with at least onetherapeutic agent. In another embodiment, the agent is a bisphosphonate.

DETAILED DESCRIPTION OF THE INVENTION A. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art.

It is noted here that as used in this specification and the appendedclaims, the singular forms “a”, “an”, and “the” also include pluralreference, unless the context clearly dictates otherwise.

The term “about” or “approximately” means within 10%, and morepreferably within 5% (or 1% or less), of a given value or range.

The terms “administer” or “administration” refer to the act of injectingor otherwise physically delivering a substance as it exists outside thebody (e.g., an antibody) into a patient, such as by mucosal,intradermal, intravenous, subcutaneous, intramuscular delivery, and/orany other method of physical delivery described herein or known in theart. When a disease, or a symptom thereof, is being treated,administration of the substance typically occurs after the onset of thedisease or symptoms thereof. When a disease or symptoms thereof, arebeing prevented, administration of the substance typically occurs beforethe onset of the disease or symptoms thereof.

An “antagonist” or “inhibitor” of TGFβ refers to a molecule that iscapable of inhibiting or otherwise decreasing one or more of thebiological activities of TGFβ, such as in a cell expressing TGFβ or in acell expressing a TGFβ ligand, or expressing a TGFβ receptor. In certainexemplary embodiments, antibodies of the invention are antagonistantibodies that inhibit or otherwise decrease the activity of TGFβ in acell having a cell surface-expressed TGFβ receptor (e.g., TGFβ receptor1, 2, or 3) when said antibody is contacted with said cell. In someembodiments, an antagonist of TGFβ (e.g., an antibody of the invention)may, for example, act by inhibiting or otherwise decreasing theactivation and/or cell signaling pathways of the cell expressing a TGFβreceptor, thereby inhibiting a TGFβ-mediated biological activity of thecell relative to the TGFβ-mediated biological activity in the absence ofantagonist. In certain embodiments of the invention, the anti-TGFβantibodies are antagonistic anti-TGFβ antibodies, preferably fullyhuman, monoclonal, antagonistic anti-TGFβ antibodies.

The terms “antibody”, “immunoglobulin”, or “Ig” may be usedinterchangeably herein. The term antibody includes, but is not limitedto, synthetic antibodies, monoclonal antibodies, recombinantly producedantibodies, multispecific antibodies (including bispecific antibodies),human antibodies, humanized antibodies, chimeric antibodies,intrabodies, single-chain Fvs (scFv) (e.g., including monospecific,bispecific, etc.), camelized antibodies, Fab fragments, F(ab′)fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id)antibodies, and epitope-binding fragments of any of the above. Inparticular, antibodies include immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules, i.e.,antigen-binding domains or molecules that contain an antigen-bindingsite that specifically binds to a TGFβ antigen (e.g., one or morecomplementarity determining regions (CDRs) of an anti-TGFβ antibody).The anti-TGFβ antibodies can be of any type (e.g., IgG, IgE, IgM, IgD,IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2),or any subclass (e.g., IgG2a and IgG2b) of an immunoglobulin molecule.In certain embodiments, the anti-TGFβ antibodies are humanized, such ashumanized monoclonal anti-TGFβ antibodies. In other embodiments, theanti-TGFβ antibodies are fully human, such as fully human monoclonalanti-TGFβ antibodies. In preferred embodiments, the anti-TGFβ antibodiesare IgG antibodies, such as IgG4 antibodies.

The terms “composition” and “formulation” are intended to encompass aproduct containing specified ingredients (e.g., an anti-TGFβ antibody)in, optionally, specified amounts, as well as any product which results,directly or indirectly, from the combination of specified ingredientsin, optionally, specified amounts.

The terms “constant region” or “constant domain” refer to a carboxyterminal portion of the light and heavy chain that is not directlyinvolved in binding of the antibody to antigen, but exhibits variouseffector functions, such as interaction with the Fc receptor. The termsrefer to the portion of an immunoglobulin molecule having a moreconserved amino acid sequence relative to the other portion of theimmunoglobulin, the variable domain, which contains the antigen-bindingsite. The constant domain contains the CH1, CH2, and CH3 domains of theheavy chain, and the CHL domain of the light chain.

The term “epitope” refers to a localized region on the surface of anantigen, such as a TGFβ polypeptide or TGFβ polypeptide fragment, thatis capable of being bound to one or more antigen-binding regions of anantibody, and that has antigenic or immunogenic activity in an animal,preferably a mammal, and most preferably in a human, that is capable ofeliciting an immune response. An epitope having immunogenic activity isa portion of a polypeptide that elicits an antibody response in ananimal. An epitope having antigenic activity is a portion of apolypeptide to which an antibody specifically binds, as determined byany method well known in the art, for example, such as an immunoassay.Antigenic epitopes need not necessarily be immunogenic. Epitopes usuallyconsist of chemically active surface groupings of molecules, such asamino acids or sugar side chains, and have specific three-dimensionalstructural characteristics, as well as specific charge characteristics.A region of a polypeptide contributing to an epitope may be contiguousamino acids of the polypeptide or the epitope may come together from twoor more non-contiguous regions of the polypeptide. The epitope may ormay not be a three-dimensional surface feature of the antigen. Incertain embodiments, a TGFβ epitope is a three-dimensional surfacefeature of a TGFβ polypeptide (e.g., in a trimeric form of a TGFβpolypeptide). In other embodiments, a TGFβ epitope is a linear featureof a TGFβ polypeptide (e.g., in a dimeric form or monomeric form of theTGFβ polypeptide). Anti-TGFβ antibodies may specifically bind to anepitope of the monomeric form of TGFβ, an epitope of the dimeric form ofTGFβ, or both the monomeric form and the dimeric form of TGFβ.

The term “excipients” refers to inert substances that are commonly usedas a diluent, vehicle, preservative, binder, stabilizing agent, etc. fordrugs and includes, but is not limited to, proteins (e.g., serumalbumin, etc.), amino acids (e.g., aspartic acid, glutamic acid, lysine,arginine, glycine, histidine, etc.), fatty acids and phospholipids(e.g., alkyl sulfonates, caprylate, etc.), surfactants (e.g., SDS,polysorbate, nonionic surfactant, etc.), saccharides (e.g., sucrose,maltose, trehalose, etc.) and polyols (e.g., mannitol, sorbitol, etc.).See, also, Remington's Pharmaceutical Sciences (1990) Mack PublishingCo., Easton, Pa., which is hereby incorporated by reference in itsentirety.

In the context of a peptide or polypeptide, the term “fragment” refersto a peptide or polypeptide that comprises less than the full lengthamino acid sequence. Such a fragment may arise, for example, from atruncation at the amino terminus, a truncation at the carboxy terminus,and/or an internal deletion of a residue(s) from the amino acidsequence. Fragments may, for example, result from alternative RNAsplicing or from in vivo protease activity. In certain embodiments, TGFβfragments include polypeptides comprising an amino acid sequence of atleast 50, at 100 amino acid residues, at least 125 contiguous amino acidresidues, at least 150 contiguous amino acid residues, at least 175contiguous amino acid residues, at least 200 contiguous amino acidresidues, or at least 250 contiguous amino acid residues of the aminoacid sequence of a TGFβ polypeptide. In a specific embodiment, afragment of a TGFβ polypeptide or an antibody that specifically binds toa TGFβ antigen retains at least 1, at least 2, or at least 3 functionsof the full-length polypeptide or antibody.

The terms “fully human antibody” or “human antibody” are usedinterchangeably herein and refer to an antibody that comprises a humanvariable region and, most preferably a human constant region. Inspecific embodiments, the terms refer to an antibody that comprises avariable region and constant region of human origin. “Fully human”anti-TGFβ antibodies, in certain embodiments, can also encompassantibodies that bind TGFβ polypeptides and are encoded by nucleic acidsequences that are naturally occurring somatic variants of a humangermline immunoglobulin nucleic acid sequence. In a specific embodiment,the anti-TGFβ antibodies are fully human antibodies. The term “fullyhuman antibody” includes antibodies having variable and constant regionscorresponding to human germline immunoglobulin sequences as described byKabat et al. (See Kabat et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242). Methods of producing fullyhuman antibodies are known in the art.

The phrase “recombinant human antibody” includes human antibodies thatare prepared, expressed, created, or isolated by recombinant means, suchas antibodies expressed using a recombinant expression vectortransfected into a host cell, antibodies isolated from a recombinant,combinatorial human antibody library, antibodies isolated from an animal(e.g., a mouse or cow) that is transgenic and/or transchromosomal forhuman immunoglobulin genes (see, e.g., Taylor, L. D. et al. (1992) NuclAcids Res. 20:6287-6295) or antibodies prepared, expressed, created, orisolated by any other means that involves splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies can have variable and constant regions derived fromhuman germline immunoglobulin sequences (See Kabat, E. A. et al. (1991)Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242).In certain embodiments, however, such recombinant human antibodies aresubjected to in vitro mutagenesis (or, when an animal transgenic forhuman Ig sequences is used, in vivo somatic mutagenesis) and thus theamino acid sequences of the VH and VL regions of the recombinantantibodies are sequences that, while derived from and related to humangermline VH and VL sequences, may not naturally exist within the humanantibody germline repertoire in vivo.

The term “heavy chain” when used in reference to an antibody refers tofive distinct types, called alpha (α), delta (Δ), epsilon (ε), gamma (γ)and mu (μ), based on the amino acid sequence of the heavy chain constantdomain. These distinct types of heavy chains are well known in the artand give rise to five classes of antibodies, IgA, IgD, IgE, IgG, andIgM, respectively, including four subclasses of IgG, namely IgG1, IgG1,IgG3, and IgG4. Preferably the heavy chain is a human heavy chain.

An “isolated” or “purified” antibody is substantially free of cellularmaterial or other contaminating proteins from the cell or tissue sourcefrom which the antibody is derived, or substantially free of chemicalprecursors or other chemicals when chemically synthesized. The language“substantially free of cellular material” includes preparations of anantibody in which the antibody is separated from cellular components ofthe cells from which it is isolated or recombinantly produced. Thus, anantibody that is substantially free of cellular material includespreparations of antibody having less than about 30%, 20%, 10%, or 5% (bydry weight) of heterologous protein (also referred to herein as a“contaminating protein”). When the antibody is recombinantly produced,it is also preferably substantially free of culture medium, i.e.,culture medium represents less than about 20%, 10%, or 5% of the volumeof the protein preparation. When the antibody is produced by chemicalsynthesis, it is preferably substantially free of chemical precursors orother chemicals, i.e., it is separated from chemical precursors or otherchemicals that are involved in the synthesis of the protein.Accordingly, such preparations of the antibody have less than about 30%,20%, 10%, 5% (by dry weight) of chemical precursors or compounds otherthan the antibody of interest. In a preferred embodiment, anti-TGFβantibodies are isolated or purified.

The terms “Kabat numbering,” and like terms are recognized in the artand refer to a system of numbering amino acid residues that are morevariable (i.e. hypervariable) than other amino acid residues in theheavy and light chain variable regions of an antibody, or anantigen-binding portion thereof (Kabat et al. (1971) Ann. NY Acad. Sci.190:382-391 and, Kabat et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242). For the heavy chainvariable region, the hypervariable region typically ranges from aminoacid positions 31 to 35 for CDR1, amino acid positions 50 to 65 forCDR2, and amino acid positions 95 to 102 for CDR3. For the light chainvariable region, the hypervariable region typically ranges from aminoacid positions 24 to 34 for CDR1, amino acid positions 50 to 56 forCDR2, and amino acid positions 89 to 97 for CDR3.

The term “light chain” when used in reference to an antibody refers totwo distinct types, called kappa (κ) of lambda (λ), based on the aminoacid sequence of the constant domains. Light chain amino acid sequencesare well known in the art. In preferred embodiments, the light chain isa human light chain.

The terms “manage”, “managing”, and “management” refer to the beneficialeffects that a subject derives from a therapy (e.g., a prophylactic ortherapeutic agent), which does not result in a cure of the disease ordisorder. In certain embodiments, a subject is administered one or moretherapies (e.g., prophylactic or therapeutic agents) to “manage” aTGFβ-mediated disease (e.g., OI), or one or more symptoms thereof, so asto prevent the progression or worsening of the disease.

The term “monoclonal antibody” refers to an antibody obtained from apopulation of homogenous or substantially homogeneous antibodies, andeach monoclonal antibody will typically recognize a single epitope onthe antigen. In preferred embodiments, a “monoclonal antibody” is anantibody produced by a single hybridoma or other cell. The term“monoclonal” is not limited to any particular method for making theantibody. For example, monoclonal antibodies may be made by thehybridoma method as described in Kohler et al.; Nature, 256:495 (1975)or may be isolated from phage libraries. Other methods for thepreparation of clonal cell lines and of monoclonal antibodies expressedthereby are well known in the art (see, for example, Chapter 11 in:Short Protocols in Molecular Biology, (2002) 5th Ed.; Ausubel et al.,eds., John Wiley and Sons, New York).

The term “pharmaceutically acceptable” means being approved by aregulatory agency of the Federal or a State government or listed in theU.S. Pharmacopeia, European Pharmacopeia, or other generally recognizedPharmacopeia for use in animals, and more particularly in humans.

The term “pharmaceutically acceptable excipient” means any inertsubstance that is combined with an active molecule, such as a monoclonalantibody, for preparing an agreeable or convenient dosage form. The“pharmaceutically acceptable excipient” is an excipient that isnon-toxic to recipients at the dosages and concentrations employed, andis compatible with other ingredients of the formulation comprising themonoclonal antibody.

The terms “prevent”, “preventing”, and “prevention” refer to the totalor partial inhibition of the development, recurrence, onset, or spreadof a TGFβ-mediated disease and/or symptom related thereto, resultingfrom the administration of a therapy or combination of therapiesprovided herein (e.g., a combination of prophylactic or therapeuticagents).

The term “TGFβ antigen” refers to that portion of a TGFβ polypeptide towhich an antibody specifically binds. A TGFβ antigen also refers to ananalog or derivative of a TGFβ polypeptide or fragment thereof to whichan antibody specifically binds. In some embodiments, a TGFβ antigen is amonomeric TGFβ antigen or a dimeric TGFβ antigen. A region of a TGFβpolypeptide contributing to an epitope may be contiguous amino acids ofthe polypeptide, or the epitope may come together from two or morenon-contiguous regions of the polypeptide. The epitope may or may not bea three-dimensional surface feature of the antigen. A localized regionon the surface of a TGFβ antigen that is capable of eliciting an immuneresponse is a TGFβ epitope. The epitope may or may not be athree-dimensional surface feature of the antigen. As used herein, an“analog” of the TGFβ antigen refers to a polypeptide that possesses asimilar or identical function as a TGFβ polypeptide, a fragment of aTGFβ polypeptide, or a TGFβ epitope described herein. For example, theanalog may comprise a sequence that is at least 80%, at least 85%, atleast 90%, at least 95%, or at least 99% identical to the amino acidsequence of a TGFβ polypeptide (e.g., SEQ ID NO: 1, 2, or 3), a fragmentof a TGFβ polypeptide, a TGFβ epitope, or an anti-TGFβ antibodydescribed herein. Additionally or alternatively, the polypeptide isencoded by a nucleotide sequence that hybridizes under stringentconditions to a nucleotide sequence encoding a TGFβ polypeptide, afragment of a TGFβ polypeptide, or a TGFβ epitope described herein

The term “human TGFβ,” “hTGFβ,” or “hTGFβ polypeptide” and similar termsrefer to the polypeptides (“polypeptides,” “peptides,” and “proteins”are used interchangeably herein) comprising the amino acid sequence ofSEQ ID NO: 1, 2, or 3, and related polypeptides, including SNP variantsthereof. Related polypeptides include allelic variants (e.g., SNPvariants); splice variants; fragments; derivatives; substitution,deletion, and insertion variants; fusion polypeptides; and interspecieshomologs, preferably, which retain TGFβ activity and/or are sufficientto generate an anti-TGFβ immune response. Also encompassed are solubleforms of TGFβ that are sufficient to generate an anti-TGFβ immunologicalresponse. As those skilled in the art will appreciate, an anti-TGFβantibody can bind to a TGFβ polypeptide, polypeptide fragment, antigen,and/or epitope, as an epitope is part of the larger antigen, which ispart of the larger polypeptide fragment, which, in turn, is part of thelarger polypeptide. hTGFβ can exist in a dimeric or monomeric form.

The terms “TGFβ-mediated disease” and “TGFβ-mediated disorder” are usedinterchangeably and refer to any disease or disorder that is completelyor partially caused by or is the result of TGFβ, e.g., hTGFβ. In certainembodiments, TGFβ is aberrantly expressed. In some embodiments, TGFβ maybe aberrantly upregulated in a particular cell type. In otherembodiments, normal, aberrant, or excessive cell signaling is caused bybinding of TGFβ to a TGFβ receptor. In certain embodiments, the TGFβreceptor (e.g., TGFβ receptor 1, 2, or 3), is expressed on the surfaceof a cell, such as an osteoblast, osteoclast, or a bone marrow stromalcell. In certain embodiments, the TGFβ-mediated disease is adegenerative bone disease, such as osteogenesis imperfecta.

The terms “specifically binds” or “specifically binding” meanspecifically binding to an antigen or a fragment thereof (e.g., TGFβ)and not specifically binding to other antigens. An antibody thatspecifically binds to an antigen may bind to other peptides orpolypeptides with lower affinity, as determined by, e.g.,radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA),BIACORE, or other assays known in the art. In certain embodiments, ananti-TGFβ antibody of the invention may specifically bind to TGFβ (e.g.,hTGFβ) with more than two-fold greater affinity that a different,non-TGFβ antigen. Antibodies or variants or fragments thereof thatspecifically bind to an antigen may be cross-reactive with relatedantigens. For example, in certain embodiments an anti-TGFβ antibody maycross-react with hTGFβ and another TGFβ antigen (e.g., a rodent ornon-human primate TGFβ antibody). Preferably, antibodies or variants orfragments thereof that specifically bind to an antigen do notcross-react with other non-TGFβ antigens. An antibody or a variant or afragment thereof that specifically binds to a TGFβ antigen can beidentified, for example, by immunoassays, BIAcore, or other techniquesknown to those of skill in the art. Typically a specific or selectivereaction will be at least twice background signal or noise, and moretypically more than 10 times background. In some embodiments, thebinding protein or antibody will bind to its antigen, e.g. TGFβ, with adissociation constant of between 1×10⁻⁶ M and 1×10⁻⁷. In otherembodiments, the dissociation constant is between 1×10⁻⁶ M and 1×10⁻⁸.See, e.g., Paul, ed., 1989, Fundamental Immunology Second Edition, RavenPress, New York at pages 332-336 for a discussion regarding antibodyspecificity.

The terms “subject” and “patient” are used interchangeably. As usedherein, a subject is preferably a mammal, such as a non-primate (e.g.,cows, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkeyand human), most preferably a human. In one embodiment, the subject is amammal, preferably a human, having a TGFβ-mediated disease. In anotherembodiment, the subject is a mammal, preferably a human, at risk ofdeveloping a TGFβ-mediated disease.

The term “therapeutic agent” refers to any agent that can be used in thetreatment, management, or amelioration of a TGFβ-mediated disease and/ora symptom related thereto. In certain embodiments, the term “therapeuticagent” refers to a TGFβ antibody. In certain other embodiments, the term“therapeutic agent” refers to an agent other than a TGFβ antibody.Preferably, a therapeutic agent is an agent that is known to be usefulfor, or has been, or is currently being used for the treatment,management, or amelioration of a TGFβ-mediated disease, or one or moresymptoms related thereto.

The term “therapy” refers to any protocol, method, and/or agent that canbe used in the prevention, management, treatment, and/or amelioration ofa TGFβ-mediated disease (e.g., OI). In certain embodiments, the terms“therapies” and “therapy” refer to a biological therapy, supportivetherapy, and/or other therapies useful in the prevention, management,treatment, and/or amelioration of a TGFβ-mediated disease known to oneof skill in the art, such as medical personnel.

The terms “treat”, “treatment”, and “treating” refer to the reduction oramelioration of the progression, severity, and/or duration of aTGFβ-mediated disease (e.g., OI) resulting from the administration ofone or more therapies (including, but not limited to, the administrationof one or more prophylactic or therapeutic agents). In specificembodiments, such terms refer to the reduction or inhibition of thebinding of TGFβ to a TGFβ receptor, the reduction or inhibition of theproduction or secretion of TGFβ from a cell expressing a TGFβ receptorof a subject, the reduction or inhibition of the production or secretionof TGFβ from a cell not expressing a TGFβ receptor of a subject, and/orthe inhibition or reduction of one or more symptoms associated with aTGFβ-mediated disease, such as OI.

The terms “variable region” or “variable domain” refer to a portion ofthe light and heavy chains, typically about the amino-terminal 120 to130 amino acids in the heavy chain and about 100 to 110 amino acids inthe light chain, which differ extensively in sequence among antibodiesand are used in the binding and specificity of each particular antibodyfor its particular antigen. The variability in sequence is concentratedin those regions called complementarity determining regions (CDRs),while the more highly conserved regions in the variable domain arecalled framework regions (FR). The CDRs of the light and heavy chainsare primarily responsible for the interaction of the antibody withantigen. Numbering of amino acid positions is according to the EU Index,as in Kabat et al. (1991) Sequences of proteins of immunologicalinterest. (U.S. Department of Health and Human Services, WashingtonD.C.) 5^(th) ed. (“Kabat et al.”). In preferred embodiments, thevariable region is a human variable region.

B. Osteogenesis Imperfecta (OI)

OI encompasses a group of congenital bone disorders characterized bydeficiencies in one or more proteins involved in bone matrix depositionor homeostasis. There are eight types of OI that are defined by theirspecific gene mutation, and the resulting protein deficiency andphenotype of the affected individual. Though phenotypes vary among OItypes, common symptoms include incomplete ossification of bones andteeth, reduced bone mass, brittle bones, and pathologic fractures.

Type-I collagen is one of the most abundant connective tissue proteinsin both calcified and non-calcified tissues. Accurate synthesis,post-translational modification, and secretion of type-I collagen arenecessary for proper tissue development, maintenance, and repair. Mostmutations identified in individuals with OI result in reduced synthesisof type-I collagen, or incorrect synthesis and/or processing of type-Icollagen.

In addition to mutations to the type-I collagen gene, other mutations ingenes that participate in the intracellular trafficking and processingof collagens have been identified in OI affected individuals. Thesegenes include molecular chaperones, such as FK506 binding protein 10(FKBP10) and heat shock protein 47 (HSP47) (Alanay et al., 2010;Christiansen et al., 2010; Kelley et al., 2011). Additional mutationshave been identified in intermolecular collagen cross-linking genes,such as procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2), andin members of the collagen prolyl hydroxylase family of genes, includingleucine proline-enriched proteoglycan (leprecan) (LEPRE1),peptidylprolyl isomerase B (cyclophilin B) (CYPB), and cartilageassociated protein (CRTAP) (Morello et al., 2006; Cabral et al., 2007;Baldridge et al., 2008; van Dijk et al., 2009; Choi et al., 2009; Barneset al., 2010; Pyott et al., 2011). Mutations aside, proteins such asbone morphogenetic protein (BMP) and transforming growth factor β (TGFβ)and their respective receptors are thought to participate in the variousOI phenotypes, though the exact mechanisms of their actions are unknown(Gebken et al., 2000).

In an embodiment, TGFβ expression is regulated by molecules that bindtype-I and type-II collagen. In certain s embodiment, a small leucinerich proteoglycan (SLRP) regulates TGFβ expression. In a specificembodiment, decorin regulates TGFβ synthesis. In a certain embodiment,decorin does not bind type-I or type-II collagen in which the3-hydroxyproline site is absent at position 986 of the type-I and/ortype-II collagen molecules.

C. Bone Biology

The vertebrate skeleton is comprised of bone, which is a living,calcified tissue that provides structure, support, protection, and asource of minerals for regulating ion transport. Bone is a specializedconnective tissue that is comprised of both cellular and acellularcomponents. The acellular extracellular matrix (ECM) contains bothcollagenous and non-collagenous proteins, both of which participate inthe calcification process. A correctly secreted and aligned ECM iscritical for proper bone formation. Pathology results when any of theECM proteins are absent, malformed or misaligned, as is evidenced inosteogenesis imperfecta.

The term “cortical bone” or “compact bone” refers to the outer layer ofbone, which is dense, rigid, and tough. The term “trabecular bone” or“cancellous bone” is the spongy inner layer of bone, which is lighterand less dense than cortical bone. The term “trabecula” refers to themicroscopic structural unit of spongy bone, which is of a rod-like shapeand collagenous composition.

Bone is a dynamic tissue that undergoes constant remodeling. The term“osteoblast” refers to a terminally-differentiated bone forming cellthat deposits osteoid. The term “osteoid” refers to immature,unmineralized bone that is comprised primarily of type-I collagen. Theterm “pre-osteoblast” refers to a proliferating immature osteoblast thatis not fully differentiated. The term “osteoprogenitor” refers to apluripotent cell that gives rise to several stromal cell types,including osteoblasts. Osteoprogenitor cells, which are commonlyreferred to as “mesenchymal stem cells,” arise in the bone marrow andcan be isolated in small numbers from circulating blood. The term“osteoclast” refers to a terminally-differentiated bone resorbing cellthat is descended from a bone marrow monocyte. Osteoclasts can beidentified by their expression of tartrate resistant acid phosphatase(TRAP).

Under normal homeostatic conditions, osteoblasts and osteoclasts work inunison to maintain bone integrity. Pathology results when bonedeposition and bone resorption become uncoupled. For example,osteopetrosis is a bone disease characterized by overly dense, hard bonethat is a result of unresorptive osteoclasts, while osteoporosis is abone disorder characterized by brittle, porous bones which can resultfrom increased osteoclast activity. Evidence suggests that osteoclastactivity may be increased in osteogenesis imperfecta, implicating thesecell types as a potential target for therapeutic intervention. Thepresent disclosure includes methods of inhibiting osteoclasts with aTGFβ antibody.

Several methods can be used to measure and characterize the structure,density, and quality of bone, including histology and histomorphometry,atomic force microscopy, confocal Raman microscopy, nanoindentation,three-point bending test, X-ray imaging, and micro computed tomography(μ-CT). In an exemplified embodiment, bones are measured andcharacterized by at least one of these methods.

The term “bone volume density” refers to the fraction of a given volumeof bone (total volume or TV) that is comprised of calcified matter (bonevolume or BV). Therefore, bone volume density is calculated as BV/TV andreported as a percentage. The term “specific bone surface” refers to thetotal bone surface (BS) per given volume of bone. Therefore, specificbone surface is calculated as BS/TV. Other common bone measurementsinclude: bone area (B.Ar), trabecular number (Tb.N); trabecular spacing(Tb.Sp); N.Oc (osteoclast number); Oc.S (osteoclast surface area);Oc.S/BS; osteoblast number (N.Ob), osteoblast surface area (Ob.S),osteoblast perimeter (Ob.Pm), and derivatives of any of saidmeasurements. A larger Oc.S/BS is an indicator of increased boneresorption by osteoclasts.

D. Transforming growth factor beta (TGFβ)

TGFβs are multifunctional cytokines that are involved in cellproliferation and differentiation, embryonic development, extracellularmatrix formation, bone development, wound healing, hematopoiesis, andimmune and inflammatory responses (Roberts et al., 1981; Border et al.,1995a). Secreted TGFβ protein is cleaved into a latency-associatedpeptide (LAP) and a mature TGFβ peptide, and is found in latent andactive forms. The mature TGFβ peptide forms both homodimers andheterodimers with other TGFβ family members. TGFβ may be purified fromany natural source, or may be produced synthetically (e.g., by use ofrecombinant DNA technology). Preferably, the TGFβ molecule is from ahuman, known herein as “hTGF.”

There are three human TGFβ isoforms: TGFβ1, TGFβ2, and TGFβ3 (Swiss Protaccession numbers P01137, P08112, and P10600, respectively) which, intheir biologically active state, are 25 kDa homodimers comprising two112 amino acid monomers joined by an inter-chain disulfide bridge. TGFβ1differs from TGFβ2 by 27, and from TGFβ3 by 22, mainly conservativeamino acid changes. These differences have been mapped on the 3Dstructure of TGFβ determined by X-ray crystallography (Schlunegger etal., 1992; Peer et al., 1996) and the receptor binding regions have beendefined (Griffith et al., 1996; Qian et al., 1996).

hTGFβ1 (SEQ ID NO: 1) (SEQ ID NO: 1)MPPSGLRLLL LLLPLLWLLV LTPGRPAAGL STCKTIDMEL VKRKRIEAIR GQILSKLRLA   60SPPSQGEVPP GPLPEAVLAL YNSTRDRVAG ESAEPEPEPE ADYYAKEVTR VLMVETHNEI  120YDKFKQSTHS IYMFFNTSEL REAVPEPVLL SRAELRLLRL KLKVEQHVEL YQKYSNNSWR  180YLSNRLLAPS DSPEWLSFDV TGVVRQWLSR GGEIEGFRLS AHCSCDSRDN TLQVDINGFT  240TGRRGDLATI HGMNRPFLLL MATPLERAQH LQSSRHRRAL DTNYCFSSTE KNCCVRQLYI  300DFRKDLGWKW IHEPKGYHAN FCLGPCPYIW SLDTQYSKVL ALYNQHNPGA SAAPCCVPQA  360LEPLPIVYYV GRKPKVEQLS NMIVRSCKCS                                   390hTGFβ2 (SEQ ID NO: 2) (SEQ ID NO: 2)MHYCVLSAFL ILHLVTVALS LSTCSTLDMD QFMRKRIEAI RGQILSKLKL TSPPEDYPEP   60EEVPPEVISI YNSTRDLLQE KASRRAAACE RERSDEEYYA KEVYKIDMPP FFPSENAIPP  120TFYRPYFRIV RFDVSAMEKN ASNLVKAEFR VFRLQNPKAR VPEQRIELYQ ILKSKDLTSP  180TQRYIDSKVV KTRAEGEWLS FDVTDAVHEW LHHKDRNLGF KISLHCPCCT FVPSNNYIIP  240NKSEELEARF AGIDGTSTYT SGDQKTIKST RKKNSGKTPH LLLMLLPSYR LESQQTNRRK  300KRALDAAYCF RNVQDNCCLR PLYIDFKRDL GWKWIHEPKG YNANFCAGAC PYLWSSDTQH  360SRVLSLYNTI NPEASASPCC VSQDLEPLTI LYYIGKTPKI EQLSNMIVKS CKCS        414hTGFβ3 (SEQ ID NO: 3) (SEQ ID NO: 3)MKMHLQRALV VLALLNFATV SLSLSTCTTL DFGHIKKKRV EAIRGQILSK LRLTSPPEPT   60VMTHVPYQVL ALYNSTRELL EEMHGEREEG CTQENTESEY YAKEIHKFDM IQGLAEHNEL  120AVCPKGITSK VFRFNVSSVE KNRTNLFRAE FRVLRVPNPS SKRNEQRIEL FQILRPDEHI  180AKQRYIGGKN LPTRGTAEWL SFDVTDTVRE WLLRRESNLG LEISIHCPCH TFQPNGDILE  240NIHEVMEIKF KGVDNEDDHG RGDLGRLKKQ KDHHNPHLIL MMIPPHRLDN PGQGGQRKKR  300ALDTNYCFRN LEENCCVRPL YIDFRQDLGW KWVHEPKGYY ANFCSGPCPY LRSADTTHST  360VLGLYNTLNP EASASPCCVP QDLEPLTILY YVGRTPKVEQ LSNMVVKSCK CS          412

There are three TGFβ receptors in humans, TGFβ receptor 1, 2, and 3,which can be distinguished by their structural and functionalproperties, including affinity for TGFβ protein family members. Bindingof a TGFβ protein to a homodimeric or heterodimeric TGFβ transmembranereceptor complex activates the canonical TGFβ signaling pathway mediatedby intracellular SMAD proteins.

The deregulation of TGFβs leads to pathological processes that, inhumans, have been implicated in numerous conditions, such as, birthdefects, cancer, chronic inflammatory, autoimmune diseases, and fibroticdiseases (Border et al., 1994; Border et al., 1995b).

Human TGFβs are very similar to mouse TGFβs: human TGFβ1 has only oneamino acid difference from mouse TGFβ1; human TGFβ2 has only three aminoacid differences from mouse TGFβ2; and human TGFβ3 is identical to mouseTGFβ3.

E. Molecules that Bind to Transforming Growth Factor Beta (TGFβ)

The present invention includes methods that comprise administering to asubject a molecule that binds to TGFβ. The TGFβ binder may be anybinding molecule, such as an antibody, a fusion protein (e.g., animmunoadhesin), an siRNA, a nucleic acid, an aptamer, a protein, or asmall molecule organic compound.

In certain embodiments, the invention includes an antibody that binds toTGFβ (an anti-TGFβ antibody), or a variant thereof, or anantigen-binding fragment thereof. Anti-TGFβ antibodies specifically bindto a TGFβ protein, polypeptide fragment, or epitope. The molecule thatbinds to TGFβ may be from any species.

In certain exemplary embodiments, the antibody that binds to TGFβ is ahumanized antibody, a fully human antibody, or a variant thereof, or anantigen-binding fragment thereof. Preferred anti-TGFβ antibodies preventbinding of TGFβ with its receptors and inhibit TGFβ biological activity(e.g., the TGFβ receptor-mediated intracellular SMAD signaling andresulting cellular activity).

In certain embodiments, the antibody, or antigen-binding fragmentthereof, is Lerdelimumab (CAT-152), Metelimumab (CAT-192), Fresolimumab(GC-1008), LY2382770, STX-100, or IMC-TR1.

In certain specific embodiments, the antibody that binds to TGFβcomprises a heavy chain variable region (VH) comprising the amino acidsequence of any one or more of the following complementarity determiningregions (CDRs):

HCDR1 (SEQ ID NO: 4) SNVIS; HCDR2 (SEQ ID NO: 5) GVIPIVDIANYAQRFKG; orHCDR3 (SEQ ID NO: 6) TLGLVLDAMDY.

In other specific embodiments, the antibody that binds to TGFβ comprisesa light chain variable region (VL) comprising the amino acid sequence ofany one or more of the following complementarity determining regions(CDRs):

LCDR1 (SEQ ID NO: 7) RASQSLGSSYLA; LCDR2 (SEQ ID NO: 8) GASSRAP; orLCDR3 (SEQ ID NO: 9) QQYADSPIT.

In a specific embodiment, the antibody that binds to TGFβ comprises aheavy chain variable region (VH) comprising the amino acid sequences ofSEQ ID NOs: 4, 5, and 6.

In another specific embodiment, the antibody that binds to TGFβcomprises a light chain variable region (VL) comprising the amino acidsequences of SEQ ID NOs: 7, 8, and 9.

In more specific embodiments, the antibody that binds to TGFβ comprisesa heavy chain variable region comprising the amino acid sequences of SEQID NOs: 4, 5, and 6; and a light chain variable region comprising theamino acid sequences of SEQ ID NOs: 7, 8, and 9.

In a specific embodiment, the antibody that binds to TGFβ comprises aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 10:

(SEQ ID NO: 10) QVQLVQSGAE VKKPGSSVKV SCKASGYTFS SNVISWVRQA PGQGLEWMGG VIPIVDIANY AQRFKGRVTI TADESTSTTY MELSSLRSED TAVYYCASTL GLVLDAMDYW GQGTLVTVSS. 

In another specific embodiment, the antibody that binds to TGFβcomprises a light chain variable region comprising the amino acidsequence of SEQ ID NO: 11:

(SEQ ID NO: 11) ETVLTQSPGT LSLSPGERAT LSCRASQSLG SSYLAWYQQK PGQAPRLLIY GASSRAPGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYADSPITFG QGTRLEIK. 

In more specific embodiments, the antibody that binds to TGFβ comprisesa heavy chain variable region comprising the amino acid sequence of SEQID NO: 10; and a light chain variable region comprising the amino acidsequence of SEQ ID NO: 11.

In some embodiments, the antibody that binds to TGFβ further comprises aconstant region, e.g., a human IgG constant region. In some embodiments,the constant region is a human IgG4 constant region. In additionalembodiments, the constant region is a modified human IgG4 constantregion. Preferably, the IgG4 constant region comprises the amino acidsequence of SEQ ID NO: 12:

(SEQ ID NO: 12) ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPSCP APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS  LSLSLGK. 

In other embodiments, the constant region is a human C_(κ) constantregion. Preferably, the C_(κ) constant region comprises the amino acidsequence of SEQ ID NO: 13:

RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQDSKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC (SEQ ID NO: 13).

In specific embodiments, the antibody that binds to TGFβ comprises aheavy chain comprising the amino acid sequence of SEQ ID NO: 14:

(SEQ ID NO: 14) QVQLVQSGAE VKKPGSSVKV SCKASGYTFS SNVISWVRQA PGQGLEWMGG VIPIVDIANY AQRFKGRVTI TADESTSTTY MELSSLRSED TAVYYCASTL GLVLDAMDYW GQGTLVTVSS ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPSCP APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS  LSLSLGK. Positions 1-120: variable region of the heavy chain (VH). The CDRs(complementarity determining regions, according to Kabat definition) areunderlined.Positions 121-447: constant region of human IgG4 (SwissProtIGHG4_HUMAN).

In other specific embodiments, the antibody that binds to TGFβ comprisesa light chain comprising the amino acid sequence of SEQ ID NO: 15:

(SEQ ID NO: 15) ETVLTQSPGT LSLSPGERAT LSCRASQSLG SSYLAWYQQK PGQAPRLLIY GASSRAPGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYADSPITFG QGTRLEIKRT VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDSK DSTYSLSSTL TLSKADYEKH KVYACEVTHQ  GLSSPVTKSF NRGEC. Positions 1-108: variable region of the light chain (VL). The CDRs(complementarity determining regions, according to Kabat definition) areunderlined.Positions 109-215: constant region of human C_(κ).

In further embodiments, the antibody that binds to TGFβ comprises aheavy chain comprising the amino acid sequence of SEQ ID NO: 14, and alight chain comprising the amino acid sequence of SEQ ID NO: 15.

In some embodiments, the antibody that binds to TGFβ is expressed by ahost cells as comprising leader sequences. The leader sequencepreferably comprises an amino acid sequence from 1-30 amino acids inlength, more preferably 25-25 amino acids, and most preferably 19 aminoacids. The heavy chain, light chain, or both the heavy and light chainmay comprise a leader sequence.

For example, the light or heavy chain leader sequence may comprise theamino acid sequence of SEQ ID NO: 16: MGWSCIILFL VATATGVHS (SEQ ID NO:16).

Accordingly, a host cell may expressing a unprocessed heavy chain maycomprise the amino acid sequence of SEQ ID NO: 17:

(SEQ ID NO: 17)MGWSCIILFL VATATGVHSQ VQLVQSGAEV KKPGSSVKVS CKASGYTFSS   50NVISWVRQAP GQGLEWMGGV IPIVDIANYA QRFKGRVTIT ADESTSTTYM  100ELSSLRSEDT AVYYCASTLG LVLDAMDYWG QGTLVTVSSA STKGPSVFPL  150APCSRSTSES TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG  200LYSLSSVVTV PSSSLGTKTY TCNVDHKPSN TKVDKRVESK YGPPCPSCPA  250PEFLGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSQEDP EVQFNWYVDG  300VEVHNAKTKP REEQFNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKGLPSS  350IEKTISKAKG QPREPQVYTL PPSQEEMTKN QVSLTCLVKG FYPSDIAVEW  400ESNGQPENNY KTTPPVLDSD GSFFLYSRLT VDKSRWQEGN VFSCSVMHEA  450LHNHYTQKSL SLSLGK,                                      466whereinpositions 1-19: leader sequencePositions 20-139: variable region of the heavy chain (VH). The CDRs(complementarity determining regions, according to Kabat definition) areunderlined.Positions 140-466: constant region of human IgG4 (SwissProtIGHG4_HUMAN).

In other exemplary embodiments, a host cell expressing a unprocessedlight chain may comprise the amino acid of SEQ ID NO: 18:

(SEQ ID NO: 18)MGWSCIILFL VATATGVHSE TVLTQSPGTL SLSPGERATL SCRASQSLGS   50SYLAWYQQKP GQAPRLLIYG ASSRAPGIPD RFSGSGSGTD FTLTISRLEP  100EDFAVYYCQQ YADSPITFGQ GTRLEIKRTV AAPSVFIFPP SDEQLKSGTA  150SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT  200LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC,                  234whereinPositions 1-19: leader sequencePositions 20-127: variable region of the light chain (VL). The CDRs(complementarity determining regions, according to Kabat definition) areunderlined.Positions 128-234: constant region of human C_(κ).

In specific embodiments, the antibody that binds to TGFβ is CAT-192 oran antigen-binding fragment thereof, and comprises a VH comprising theamino acid sequence of SEQ ID NO: 22 and a VL comprising the amino acidsequence of SEQ ID NO: 23:

(SEQ ID NO: 22) EVQLVESGGG VVQPGRSLRL SCAASGFTFS SYGMHWVRQA PGKELEWVAV ISYDGSIKYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARTG EYSGYDTDPQ YSWGQGTTVT  VSS  (SEQ ID NO: 23)EIVLTQSPSS LSASVGDRVT ITCRASQGIG DDLGWYQQKP GKAPILLIYG TSTLQSGVPS RFSGSGSGTD FTLTINSLQP EDFATYYCLQ DSNYPLTFGG GTRLEIK. 

In specific embodiments, the antibody that binds to TGFβ is CAT-152 oran antigen-binding fragment thereof, and comprises a VH comprising theamino acid sequence of SEQ ID NO: 24 and a VL comprising the amino acidsequence of SEQ ID NO: 25:

(SEQ ID NO: 24) EVQLVESGGG VVQPGRSLRL SCAASGFTFS SYGMHWVRQA PGKGLEWVAV IWYDGSNKYY ADSVKGRFTI SRDNSKNTLY LQMDSLRAED TAVYYCGRTL ESSLWGQGTL VTVSS  (SEQ ID NO: 25)SSELTQDPAV SVALGQTVRI TCQGDSLRSY YASWYQQKPG QAPVLVIYGK NNRPSGIPDR FSGSSSGNTA SLTITGAQAE DEADYYCNSR DSSSTHRGVF GGGTKLTVLG. 

In an exemplary embodiment of the invention, the antibody that binds toTGFβ is a humanized or fully human antibody. Examples of humanized andfully human antibody isotypes include IgA, IgD, IgE, IgG, and IgM.Preferably, the anti-TGFβ antibody is an IgG antibody. There are fourforms of IgG. Preferably, the anti-TGFβ antibody is an IgG4 antibody. Inone embodiment of the invention, the anti-TGFβ antibody is a humanizedIgG4 antibody. In another embodiment of the invention, the anti-TGFβantibody is a fully human IgG4 antibody.

In a most preferred embodiment of the invention, the anti-TGFβ antibodyis an IgG4 anti-TGFβ antibody comprising a heavy chain comprising theamino acid sequence of SEQ ID NO: 14 and a light chain comprising theamino acid sequence of SEQ ID NO: 15. In an alternative most preferredembodiment of the invention, the anti-TGFβ antibody is an IgG4 anti-TGFβantibody comprising a heavy chain variable region and a light chainvariable region, the heavy chain variable region comprising 3complementarity determining regions (CDRs) comprising the amino acidsequences of SEQ ID NOs: 4, 5, and 6, and the light chain variableregion comprising 3 CDRs comprising the amino acid sequences of SEQ IDNOs: 7, 8, and 9. Identification, isolation, preparation, andcharacterization of anti-TGFβ antibodies, including the anti-TGFβantibody comprising a heavy chain amino acid sequence comprising SEQ IDNO: 14 and a light chain amino acid sequence comprising SEQ ID NO: 15,and the CDR sequences corresponding with SEQ ID NOs: 4-9 have beendescribed in detail in U.S. Pat. Nos. 7,723,486, and 8,383,780, each ofwhich is incorporated herein by reference in its entirety.

Preferably, the antibody or antigen-binding fragment thereof is“pan-specific” and binds to human TGFβ1, TGFβ2, and TGFβ3. Morepreferably, the antibody or antigen-binding fragment thereof binds tohuman TGFβ1, TGFβ2, and TGFβ3, and acts as an antagonist. Mostpreferably, the antibody or antigen-binding fragment thereof binds tohuman TGFβ1, TGFβ2, and TGFβ3, and neutralizes human TGFβ1, TGFβ2, andTGFβ3. Exemplary pan-specific anti-TGFβ monoclonal antibodies (mAbs)suitable for use in the methods of the invention are described in U.S.Pat. Nos. 7,723,476 and 8,383,780, each of which is incorporated byreference herein in its entirety.

1D11.16 is an exemplary murine pan-specific anti-TGFβ antibody thatneutralizes human and mouse TGFβ1, TGFβ2, and TGFβ3 in a wide range ofin vitro assays (Dasch et al., 1989; Dasch et al., 1996; R&D Systemproduct sheet for MAB1835, each of which is incorporated herein byreference in their entirety) and is efficacious in proof-of principlestudies in animal models of fibrosis (Ling et al., 2003; Miyajima etal., 2000; Schneider et al., 1999; Khanna et al., 1999; Shenkar et al.,1994). However, since 1D11.16 is a murine monoclonal antibody (Dasch etal., 1989; Dasch et al., 1996), it is not a preferred for therapeuticuse in humans. Accordingly, in certain embodiments, variants orderivatives of the 1D11.16 antibody are employed in the methods of theinvention.

As indicated above, certain embodiments of the invention also includevariants or derivatives of anti-TGFβ antibodies. Specifically, theinvention may include variants of the anti-TGFβ antibody that is an IgG4anti-TGFβ antibody comprising a heavy chain comprising the amino acidsequence of SEQ ID NO: 14, and a light chain comprising the amino acidsequence of SEQ ID NO: 15. In other embodiment, the invention includesvariants or derivatives of the 1D11.16 antibody. Variants of anti-TGFβantibodies may have similar physicochemical properties based on theirhigh similarity, and therefore are also included within the scope of theinvention. Variants are defined as antibodies with an amino acidsequence that is at least 80%, at least 90%, at least 95%, or at least97%, e.g., least 98% or 99% homologous to an anti-TGFβ antibodydescribed herein, and capable of competing for binding to a TGFβpolypeptide, a TGFβ polypeptide fragment, or a TGFβ epitope. Preferably,the variants will ameliorate, neutralize, or otherwise inhibit bindingof TGFβ with its receptors and TGFβ biological activity (e.g., TGFβreceptor-mediated intracellular SMAD signaling and resulting cellularactivity). Determining competition for binding to the target can be doneby routine methods known to the skilled person in the art. Preferablythe variants are human antibodies, and preferably are IgG4 molecules. Inpreferred embodiments, a variant is at least 90%, 95%, 96%, 97%, 98%, or99% identical in amino acid sequence with the IgG4 anti-TGFβ antibodycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO: 14, and a light chain comprising the amino acid sequence of SEQ IDNO: 15. The term “variant” refers to an antibody that comprises an aminoacid sequence that is altered by one or more amino acids compared to theamino acid sequences of the anti-TGFβ antibody. The variant may haveconservative sequence modifications, including amino acid substitutions,modifications, additions, and deletions.

Examples of modifications include, but are not limited to,glycosylation, acetylation, pegylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, and linkage to a cellular ligand or other protein. Amino acidmodifications can be introduced by standard techniques known in the art,such as site-directed mutagenesis, molecular cloning,oligonucleotide-directed mutagenesis, and random PCR-mediatedmutagenesis in the nucleic acid encoding the antibodies. Conservativeamino acid substitutions include the ones in which the amino acidresidue is replaced with an amino acid residue having similar structuralor chemical properties. Families of amino acid residues having similarside chains have been defined in the art. These families include aminoacids with basic side chains (e.g., lysine, arginine, histidine), acidicside chains (e.g., aspartic acid, glutamic acid), uncharged polar sidechains (e.g., asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., glycine, alanine,valine, leucine, isoleucine, proline, phenylalanine, methionine),beta-branched side chains (e.g., threonine, valine, isoleucine), andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan). Itwill be clear to the skilled artisan that classifications of amino acidresidue families other than the one used above can also be employed.Furthermore, a variant may have non-conservative amino acidsubstitutions, e.g., replacement of an amino acid with an amino acidresidue having different structural or chemical properties. Similarminor variations may also include amino acid deletions or insertions, orboth. Guidance in determining which amino acid residues may besubstituted, modified, inserted, or deleted without abolishingimmunological activity may be found using computer programs well knownin the art. Computer algorithms, such as, inter alia, Gap or Bestfit,which are known to a person skilled in the art, can be used to optimallyalign amino acid sequences to be compared and to define similar oridentical amino acid residues. Variants may have the same or different,either higher or lower, binding affinities compared to an anti-TGFβantibody, but are still capable of specifically binding to TGFβ, and mayhave the same, higher or lower, biological activity as the anti-TGFβantibody.

Embodiments of the invention also include antigen-binding fragments ofthe anti-TGFβ antibodies. The term “antigen-binding domain,”“antigen-binding region,” “antigen-binding fragment,” and similar termsrefer to that portion of an antibody that comprises the amino acidresidues that interact with an antigen and confer on the binding agentits specificity and affinity for the antigen (e.g., the complementaritydetermining regions (CDR)). The antigen-binding region can be derivedfrom any animal species, such as rodents (e.g., rabbit, rat or hamster)and humans. Preferably, the antigen-binding region will be of humanorigin. Non-limiting examples of antigen-binding fragments include: Fabfragments, F(ab′)2 fragments, Fd fragments, Fv fragments, single chainFv (scFv) molecules, dAb fragments, and minimal recognition unitsconsisting of the amino acid residues that mimic the hypervariableregion of the antibody.

F. Therapeutic Administration

The methods described herein comprise administering a therapeuticallyeffective amount of an antibody that binds to TGFβ to a subject. As usedherein, the phrase “therapeutically effective amount” means a dose ofantibody that binds to TGFβ that results in a detectable improvement inone or more symptoms associated with OI or which causes a biologicaleffect (e.g., a decrease in the level of a particular biomarker) that iscorrelated with the underlying pathologic mechanism(s) giving rise tothe condition or symptom(s) of osteogenesis imperfecta. For example, adose of antibody that binds to TGFβ that increases bone mineral density,increases bone mass and/or bone strength, reduces bone and/or toothfractures, and/or improves any diagnostic measurement of OI is deemed atherapeutically effective amount.

In an embodiment, bone mineral density, bone mass, and/or bone strengthare increased by about 5% to about 200% following treatment with anantibody that binds to TGFβ. In certain embodiments, bone mineraldensity, bone mass, and/or bone strength are increased by about 5% toabout 10%, 10% to about 15%, 15% to about 20%, 20% to about 25%, 25% toabout 30%, 30% to about 35%, 35% to about 40%, 40% to about 45%, 45% toabout 50%, 50% to about 55%, 55% to about 60%, 60% to about 65%, 65% toabout 70%, 70% to about 75%, 75% to about 80%, 80% to about 85%, 85% toabout 90%, 90% to about 95%, 95% to about 100%, 100% to about 105%, 105%to about 110%, 110% to about 115%, 115% to about 120%, 120% to about125%, 125% to about 130%, 130% to about 135%, 135% to about 140%, 140%to about 145%, 145% to about 150%, 150% to about 155%, 155% to about160%, 160% to about 165%, 165% to about 170%, 170% to about 175%, 175%to about 180%, 180% to about 185%, 185% to about 190%, 190% to about195%, or 195% to about 200%, following treatment with an antibody thatbinds to TGFβ.

In certain embodiments, a dose of an antibody which reduces serumbiomarkers of bone resorption, such as urinary hydroxyproline, urinarytotal pyridinoline (PYD), urinary free deoxypyridinoline (DPD), urinarycollagen type-I cross-linked N-telopeptide (NTX), urinary or serumcollagen type-I cross-linked C-telopeptide (CTX), bone sialoprotein(BSP), osteopontin (OPN), and tartrate-resistant acid phosphatase 5b(TRAP), is deemed a therapeutically effective amount. In an embodiment,serum biomarkers of bone resorption are reduced by about 5% to about200% following treatment with an antibody that binds to TGFβ

In an embodiment, serum biomarkers of bone resorption, such as urinaryhydroxyproline, urinary total pyridinoline (PYD), urinary freedeoxypyridinoline (DPD), urinary collagen type-I cross-linkedN-telopeptide (NTX), urinary or serum collagen type-I cross-linkedC-telopeptide (CTX), bone sialoprotein (BSP), osteopontin (OPN), andtartrate-resistant acid phosphatase 5b (TRAP), are decreased by about 5%to about 10%, 10% to about 15%, 15% to about 20%, 20% to about 25%, 25%to about 30%, 30% to about 35%, 35% to about 40%, 40% to about 45%, 45%to about 50%, 50% to about 55%, 55% to about 60%, 60% to about 65%, 65%to about 70%, 70% to about 75%, 75% to about 80%, 80% to about 85%, 85%to about 90%, 90% to about 95%, 95% to about 100%, 100% to about 105%,105% to about 110%, 110% to about 115%, 115% to about 120%, 120% toabout 125%, 125% to about 130%, 130% to about 135%, 135% to about 140%,140% to about 145%, 145% to about 150%, 150% to about 155%, 155% toabout 160%, 160% to about 165%, 165% to about 170%, 170% to about 175%,175% to about 180%, 180% to about 185%, 185% to about 190%, 190% toabout 195%, or 195% to about 200%, following treatment with an antibodythat binds to TGFβ.

In certain embodiments, a dose of an antibody which increase serumbiomarkers of bone deposition, such as total alkaline phosphatase,bone-specific alkaline phosphatase, osteocalcin, and type-I procollagen(C-terminal/N-terminal), is deemed a therapeutically effective amount.In an embodiment, serum biomarkers of bone deposition are increased byabout 5% to about 200% following treatment with an antibody that bindsto TGFβ.

In an embodiment, serum biomarkers of bone deposition, such as totalalkaline phosphatase, bone-specific alkaline phosphatase, osteocalcin,and type-I procollagen (C-terminal/N-terminal), are increased by about5% to about 10%, 10% to about 15%, 15% to about 20%, 20% to about 25%,25% to about 30%, 30% to about 35%, 35% to about 40%, 40% to about 45%,45% to about 50%, 50% to about 55%, 55% to about 60%, 60% to about 65%,65% to about 70%, 70% to about 75%, 75% to about 80%, 80% to about 85%,85% to about 90%, 90% to about 95%, 95% to about 100%, 100% to about105%, 105% to about 110%, 110% to about 115%, 115% to about 120%, 120%to about 125%, 125% to about 130%, 130% to about 135%, 135% to about140%, 140% to about 145%, 145% to about 150%, 150% to about 155%, 155%to about 160%, 160% to about 165%, 165% to about 170%, 170% to about175%, 175% to about 180%, 180% to about 185%, 185% to about 190%, 190%to about 195%, or 195% to about 200%, following treatment with anantibody that binds to TGFβ.

Other embodiments include administering a therapeutically effective doseof an antibody which improves the function of non-skeletal organsaffected by OI. For example, a dose of antibody that binds to TGFβ thatimproves hearing, lung, and/or kidney function is deemed atherapeutically effective amount.

In accordance with the methods of the present invention, atherapeutically effective amount of an antibody that binds to TGFβ thatis administered to a subject will vary depending upon the age and thesize (e.g., body weight or body surface area) of the subject, as well asthe route of administration, and other factors well known to those ofordinary skill in the art.

In certain exemplary embodiments, the anti-TGFβ antibody is administeredto the subject as a subcutaneous dose. Other exemplary modes ofadministration include, but are not limited to, intradermal,intramuscular, intraperitoneal, intravenous, intranasal, epidural, andoral routes. The composition may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local. TheTGFβ antibody can be administered parenterally or subcutaneously.

Various delivery systems are known and can be used to administer thepharmaceutical composition, e.g., encapsulation in liposomes,microparticles, microcapsules, receptor mediated endocytosis (see, e.g.,Wu et al. (1987) J. Biol. Chem. 262:4429-4432). The therapeuticcompositions will be administered with suitable carriers, excipients,and other agents that are incorporated into formulations to provideimproved transfer, delivery, tolerance, and the like. A multitude ofappropriate formulations can be found in the formulary known to allpharmaceutical chemists: Remington's Pharmaceutical Sciences, MackPublishing Company, Easton, Pa. These formulations include, for example,powders, pastes, ointments, jellies, waxes, oils, lipids, lipid(cationic or anionic) containing vesicles (such as LIPOFECTIN™), DNAconjugates, anhydrous absorption pastes, oil-in-water and water-in-oilemulsions, emulsions carbowax (polyethylene glycols of various molecularweights), semi-solid gels, and semi-solid mixtures containing carbowax.See also Powell et al. “Compendium of excipients for parenteralformulations” PDA (1998) J Pharm Sci Technol 52:238-311.

Pharmaceutical compositions may be prepared into dosage forms in a unitdose suited to fit a dose of the active ingredients. Such dosage formsin a unit dose include, for example, tablets, pills, capsules,injections (ampoules), suppositories, etc.

Pharmaceutical compositions can also be administered to the subjectusing any acceptable device or mechanism. For example, theadministration can be accomplished using a syringe and needle or with areusable pen and/or autoinjector delivery device. The methods of thepresent invention include the use of numerous reusable pen and/orautoinjector delivery devices to administer a TGFβ binder (orpharmaceutical formulation comprising the binder). Examples of suchdevices include, but are not limited to AUTOPEN™ (Owen Mumford, Inc.,Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf,Switzerland), HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen(Eli Lilly and Co., Indianapolis, Ind.), NOVOPEN™ I, II and III (NovoNordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk,Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, N.J.),OPTIPEN™, OPTIPEN PRO™ OPTIPEN STARLET™, and OPTICLIK™ (sanofi-aventis,Frankfurt, Germany), to name only a few. Examples of disposable penand/or autoinjector delivery devices having applications in subcutaneousdelivery of a pharmaceutical composition include, but are not limited tothe SOLOSTAR™ pen (sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and theKWIKPEN™ (Eli Lilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks,Calif.), the PENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey,L. P.), and the HUMIRA™ Pen (Abbott Labs, Abbott Park, Ill.), to nameonly a few.

The use of a microinfusor to deliver a TGFβ binder (or pharmaceuticalformulation comprising the binder) to a subject is also contemplatedherein. As used herein, the term “microinfusor” means a subcutaneousdelivery device designed to slowly administer large volumes (e.g., up toabout 2.5 mL or more) of a therapeutic formulation over a prolongedperiod of time (e.g., about 10, 15, 20, 25, 30 or more minutes). See,e.g., U.S. Pat. Nos. 6,629,949; 6,659,982; and Meehan et al., J.Controlled Release 46:107-116 (1996). Microinfusors are particularlyuseful for the delivery of large doses of therapeutic proteins containedwithin high concentration (e.g., about 100, 125, 150, 175, 200 or moremg/mL) and/or viscous solutions. G. Combination Therapies

In certain aspects, the invention includes methods for treating OI thatcomprise administering to a subject in need of such treatment anantibody that binds to TGFβ in combination with at least one additionaltherapeutic agent. Examples of additional therapeutic agents that can beadministered in combination with an anti-TGFβ antibody in the practiceof the methods of the present invention include, but are not limited to,bisphosphonates, calcitonin, teriparatide, and any other compound knownto treat, prevent, or ameliorate osteogenesis imperfecta in a subject.In the present methods, the additional therapeutic agent(s) can beadministered concurrently or sequentially with the antibody that bindsto TGFβ. For example, for concurrent administration, a pharmaceuticalformulation can be made that contains both an antibody that binds toTGFβ and at least one additional therapeutic agent. In an embodiment,the antibody that binds to TGFβ is administered in combination withpharmaceutical bisphosphonates (e.g., Etidronate, Clodronate,Tiludronate, Pamidronate, Neridronate, Olpadronate, Alendronate,Ibandronate, Zoledronate, and Risedronate). In another embodiment, theantibody that binds to TGFβ is administered in combination with a drugthat stimulates bone formation, such as parathyroid hormone analogs andcalcitonin. In yet another embodiment, the antibody that binds to TGFβis administered in combination with a selective estrogen receptormodulator (SERM). The amount of the additional therapeutic agent that isadministered in combination with the antibody that binds to TGFβ in thepractice of the methods of the present invention can be easilydetermined using routine methods known and readily available in the art.

Examples

OI is a generalized connective tissue disease in which affectedindividuals display an abnormality in forming type-I collagen fibrilsdue to mutations in the primary sequence of the alpha 1 or alpha 2 chainof type-I collagen, as well abnormalities in the post-translationalmodification of type-I collagen and proteins that bind type-I collagenfibrils. CRTAP encodes a protein called the cartilage associatedprotein, a member of the prolyl-3-hydroxylation complex whose functionis to assist with the proper folding, post-translational modificationand secretion of type-I collagen. Mutations to CRTAP are responsible fortype VII osteogenesis imperfecta. Mice lacking the Crtap gene(Crtap^(−/−)) display a phenotype that mimics osteogenesis imperfecta,and are used as a model of this disease (Morello et al., 2006).Crtap^(−/−) mice and age-matched wild type (WT) littermate controls wereused in the following examples.

Material and Methods Animals, Anti-TGFβ Treatment and Tissue Collection

Crtap^(−/−) mice were generated and maintained on a mixedC57Black/6J/129Sv genetic background. Mice harboring a G610C mutation inthe Col1a2 gene (Col1a2^(tm1.1Mcbr)) were obtained and bred to wildtypeC57B1/6J mice. Mice that were heterozygous for the Col1a2^(tm1.1Mcbr)allele were used for experiments. TGFβ-reporter mice that expressluciferase in response to the Smad2/3 dependent TGFβ signaling pathway(SBE-Luc mice) were obtained and bred to Crtap^(+/−) mice for 2generations to generate Crtap^(−/−) mice and wildtype littermatesexpressing the reporter transgene. All mice were housed in a vivariumand animal experiments were performed following the approved protocol ofthe Animal Care and Use Committee (IACUC).

For protein and RNA analyses, calvaria of P3 mice were isolated, cleanedof extraskeletal tissue, and snap frozen in liquid nitrogen. Forimmunostaining of Crtap^(−/−) P10 mice lungs, the lungs of each mousewere equally inflated immediately after sacrifice by gravity with 4%paraformaldehyde at a constant pressure of 25 cm H₂O and then sutureclosed at the trachea. Lungs were then gently dissected from the thoraxand fixed in 4% paraformaldehyde overnight.

Eight week old female Crtap^(−/−) and Col1a2^(tm1.1Mckbr) mice weretreated with the pan-TGFβ neutralizing antibody 1D11 for 8 weeks (10mg/kg body weight, I.P. injections 3 times each week). ControlCrtap^(−/−), Col1a2^(tm1.1Mcbr), and WT mice received a control antibody(13C4) of the same IgG1 isotype. After treatment, mice were sacrificed,and lumbar spines and femurs were collected and fixed in 10% formalinfor microCT and bone histomorphometry. Contralateral femurs ofCrtap^(−/−) mice were stored in −20° C. wrapped in saline soaked gauzeuntil biomechanical testing was performed. Lungs of these Crtap^(−/−)mice were equally inflated, collected and fixed as described for P10mice. No blinding was possible during treatment, because 1D11 or controlantibody were injected according to group allocation. In all subsequentanalyses the investigators were blinded to genotype and treatment group.

Immunoblotting

Protein was extracted from snap frozen P3 calvaria samples, transferredto 300 μl lysis buffer (0.0625 M Tris-HCl pH 7.5, 2% SDS, 5 mM NaF, 2 mMNa₃VO₄ and RocheComplete proteinase inhibitor) and homogenized for 1minute, followed by incubation at 95° C. for 40 minutes. The supernatantwas transferred to Centrifugal Filter Units/Amicon Ultra 3K (Millipore)and centrifuged to concentrate the protein. The total proteinconcentration of the lysate was measured using the Micro BCA reagent(Pierce) following the manufacturer's directions. 40 μg of calvariaprotein extracts were suspended in laemmeli buffer containing 5%β-mercaptoethanol and separated on Mini Protean TGX SDS-PAGE gels(gradient 4-20%; Bio-Rad) and transferred onto PVDF membranes forwestern blot analyses. PVDF membranes were incubated with pSmad2monoclonal antibody (Cell Signaling #3108, 1:750 in TBST containing 5%BSA overnight), followed by secondary HRP-linked anti-rabbit antibody(GE, 1:5000 in TBST containing 5% BSA for 2 hours), treated with ECLPlus Western Blotting Detection System (GE) and exposed to X-ray film.Subsequently, antibodies were stripped from membranes using ReBlot Plusreagent (Millipore), and incubated with Smad2 monoclonal antibody (CellSignaling #5339, 1:2000 in TBST containing 5% BSA overnight), followedby similar secondary antibody incubation and ECL mediated visualization.X-ray films were scanned and the density of each band was quantifiedusing ImageJ software (National Institutes of Health).

Quantitative Real-Time PCR

Total RNA was extracted from snap frozen P3 mouse calvaria using Trizolreagent (Invitrogen). The Superscript III RT system (Invitrogen) wasused to synthesize cDNA from total RNA according to the manufacturer'sprotocol. Quantitative RT-PCR was performed on a LightCycler.v 1.5(Roche) using gene-specific primers and SYBR Green I reagent (Roche).β2-Microglobulin was used as the reference gene for normalizing cDNAconcentrations.

In Vivo Bioluminescence Imaging

P10 Ctrap^(−/−) mice and wildtype littermates that expressed theTGFβ-reporter transgene (SBE-Luc mice) were injected with D-luciferin(Goldbio, 150 mg/kg, IP), anaesthetized with isoflurane, and imaged 10minutes after injection using a bioluminescence imaging system(Xenogen).

Primary Osteoblast Culture, TGFβ-Reporter Cells

Bone marrow cells were isolated from tibias and femurs of approximately2 month old Crtap^(−/−) and wildtype mice and cultured in α-MEM suppliedwith 10% FBS, 100 U/mL penicillin and 100 ug/mL streptomycin. Media waschanged every second day and unattached cells were discarded. After 7days, the attached cells, defined as bone marrow stromal cells (BMSCs),were reseeded to 24-well plates at 2.5×10⁴ cells per cm² and cultured inosteogenic medium (α-MEM, 10% FBS, 500 μM ascorbic acid, and 10 mMβ-glycerophosphate) for 3 days. Conditioned medium was collected andincubated with PAI-luciferase reporter mink lung epithelial cells. After24 hours, the cell lysates were collected for luciferase activityassays, which were measured using the Dual-Luciferase Reporter System(Promega). The results were normalized to the total protein amountquantified using the Micro BCA reagent (Pierce).

MicroCT, Bone Histomorphometry

Lumbar vertebrae and femurs were scanned using a Scanco μCT-40 microCTfor quantification of trabecular and cortical bone parameters. Vertebraland femoral trabecular bone parameters were analyzed using the Scancoanalysis software by manually contouring the trabecular bone ofvertebral body L4 as well as the distal metaphyseal section of thefemur. The cortical bone parameters at the center of the femoralmidshaft were quantified using the automated thresholding algorithmincluded in the software.

Scanned undecalcified Crtap^(−/−) mouse spine samples were then embeddedin plastic for sectioning. Toluidine blue staining and TRAP staining wasperformed using standard protocols for visualization and quantificationof Ob's and Oc's, respectively, using the Bioquant Osteo Image AnalysisSystem.

Immunostaining and Histology

For immunohistochemistry, hind limbs of P5 mice were collected, fixedovernight in 4% paraformaldehyde and embedded in paraffin. Afterdeparaffinization and rehydration, heat-induced antigen retrieval wasperformed (Dako, S1700) followed by treatment with hyaluronidase for 30min (2 mg/ml; Sigma). Endogenous peroxidase was blocked using 3%hydrogen peroxide for 10 min. After incubation with blocking solution(3% normal goat serum, 0.1% BSA, 0.1% Triton X-100 in PBS), sectionswere incubated in antibodies for TGFβ1 (G1221, Promega) and decorin(LF-113, kindly provided from Larry Fisher, National Institute of Dentaland Craniofacial Research, Bethesda, Md., USA) for 60 min (1:25 dilutioneach in PBS, control samples were incubated in PBS only) at 37C, andsubsequently incubated with secondary antibody (SuperPicTure PloymerDetection kit, Invitrogen). Substrate DAB was added according to themanufacturer's recommendations and samples were dehydrated and mountedusing Cytoseal XYL xylene based mounting medium (Thermo Scientific).Sections of WT and mutant littermates were processed at the same time.Images of the trabecular bone were taken with a light microscope(Axioplan 2, Zeiss) using identical exposure times for WT and mutantlittermates.

Lungs of P10 and 16 week old Crtap^(−/−) mice were equally inflatedduring tissue collection, fixed in 4% paraformaldehyde, and wereparaffin embedded. Lungs of P10 Crtap^(−/−) and wildtype mice were usedfor immunostaining for pSmad2. Briefly, paraffin sections were treatedwith xylene, rehydrated, and heated for 20 minutes for antigen retrieval(pH 6; Dako). Sections were then incubated in blocking solution (3%normal Donkey serum, 0.1% BSA, 0.1% Triton X-100 in PBS), andsubsequently incubated with rabbit anti-pSmad2 antibody (1:500) (Cellsignaling, #3108), donkey anti-rabbit secondary antibody conjugated toAlexa flour 594 (1:600) (Invitrogen), and mounted with Prolong Goldanti-fade reagent with DAPI (Invitrogen). Fluorescent images from thesesections were taken using a Zeiss microscope (Axiovision Software) usingidentical exposure times.

For lung histology and morphometry of 16 week old mice, parasagittalsections were stained using a standard protocol for Hematoxylin andEosin staining. The mean linear intercept (MLI) method was used toquantify the distance between alveolar structures. Briefly, 10histological fields were captured per mouse at 20× magnification fromall lobes of both lungs using a light microscope (Axioplan 2, Zeiss).The MLI was measured using modified ImageJ software (National Institutesof Health, modified by Paul Thompson). After manual removal of bloodvessels, large airways and other nonalveolar structures, the softwareautomatically thresholds the alveolar tissue in each image and overlaysa line grid comprised of 1,353 lines with each line measuring 21 pixelsover the image. The number of lines that intercepted alveolar structureswas used to calculate the MLI.

Biomechanical Testing by 3-Point Bending

Crtap^(−/−) and WT femurs were tested by three point bending using aspan of 6 mm with an Instron 5848 device (Instron Inc., Norwood Mass.).All the femurs were tested wet at room temperature. They were preloadedto 1 N at a rate of 0.05 N/s for 5 seconds. Following the pre-loading,the femurs were loaded to failure at a rate of 0.1 mm/sec. Load anddisplacement data was captured at rate of 40 Hz by using BLUEHILLSoftware (Instron 5848).

To determine the Yield Point, a region was identified after the preloadand before the maximum load on the load-displacement curve. This regionwas separated into 5 segments from which the fitted line of the segmentwith greatest slope was taken. Next, a 0.012 mm offset was implementedon the line. The point of intersection between the offset line and theload-displacement curve was the 0.012 Offset Yield Point. This yieldpoint corresponded more closely to a 0.2% offset strain, which iscommonly chosen in the literature. The elastic region was identified asthe region from the completion of the preload to the Yield Point. ThePost-Yield region was identified as the region from the Yield Pointuntil the point at which the change in load exceeded −1N, indicatingfailure. The Elastic Displacement was the displacement during whichspecimen remained in the elastic region. The Post-Yield Displacement wasthe displacement during which the specimen remained in the Post-Yieldregion. The Total Displacement was calculated as the sum of the ElasticDisplacement and the Post-Yield Displacement. Using the trapezoidalnumerical integration method, Energy to failure was calculated as thearea under the Load-Displacement curve. The Maximum Load was determinedby finding the highest load value recorded by BLUEHILL, before thespecimen failed. To calculate Stiffness, the Least Square fit method wasapplied to the steepest segment of the elastic region of theload-displacement curve. Stiffness was the slope of least square fitline. Geometric data (diameter and moment of inertia) obtained frommicroCT analysis of the femoral midshaft were utilized to calculate theintrinsic material properties: ultimate strength, toughness to failureand elastic modulus.

Serum Bone Turnover Markers

Serum osteocalcin (OCN) was quantified using the Mouse Osteocalcin EIAKit from Biomedical Technologies Inc. C-terminal cross-linkedtelopeptide of bone collagen (CTX) was quantified using the RatLaps™ EIAKit from Immunodiagnostic Systems Ltd. Both analyses were performedaccording to the manufacturer's protocols.

Collagen SDS-PAGE, Mass Spectrometry and Crosslinks Analyses

For mass spectrometry, type I collagen was prepared from Crtap^(−/−) andwildtype tibiae. Bone was defatted with chloroform/methanol (3:1 v/v)and demineralized in 0.5 M EDTA, 0.05 M Tris-HCl, pH 7.5, all steps at4° C. Bone were finely minced and collagen solubilized by heatdenaturation (90° C.) in SDS-PAGE sample buffer. Collagen α-chains werecut from SDS-PAGE gels and subjected to in-gel trypsin digestion.Electrospray MS was performed on the tryptic peptides using an LCQ DecaXP ion-trap mass spectrometer equipped with in-line liquidchromatography (LC) (ThermoFinnigan) using a C8 capillary column (300μm×150 mm; Grace Vydac 208 MS5.315) eluted at 4.5 μl min. Sequest searchsoftware (ThermoFinnigan) was used for peptide identification using theNCBI protein database.

Pyridinoline cross-links (HP and LP) were quantified by HPLC afterhydrolyzing demineralized bone in 6N HCl.

Surface Plasmon Resonance Analysis

Surface plasmon resonance experiments were carried out using a BIACore Xinstrument (GE Healthcare Bio-Science Corp.). Purified native mousetendon type I collagen from wild type and Crtap^(−/−) mice wereimmobilized on a CM5 sensor chip by amide coupling at a concentration ofabout 0.05 ng/mm² (500 RU) and 0.08 ng/mm² (800 RU), respectively. Theexperiments were conducted at a flow rate of 10 μl/min and 20° C. inHBS-P buffer (10 mM Hepes buffer, pH 7.4, containing 150 mM NaCl and0.005% Surfactant P20). Recombinant human decorin core protein (R&Dsystems) was injected onto both type I CM5 chips. The concentration ofthe stock solution of human decorin was determined by amino acidanalysis. The binding response of decorin to wild type and Crtap^(−/−)mouse type I collagen was normalized by the amounts of immobilized typeI collagen on the CM5 sensor chips. Three concentrations of decorin wereused (3, 5 and 12 μM), for each concentration the analysis was repeatedthree times. This experiment was performed twice with collagen isolatedfrom different mice each time

Statistical Methods

Comparisons between two groups were performed using unpaired, two-tailedStudent's t-tests. For comparisons between 3 groups, One Way Analysis ofVariance (ANOVA) was performed if equal variance of groups wasconfirmed, followed by all pairwise multiple comparison using theHolm-Sidak method. If the equal variance test failed, Kruskal-Wallis OneWay ANOVA on Ranks was performed, followed by all pairwise multiplecomparison using the Tukey Test. A P value less than 0.05 was consideredstatistically significant for Student's t-test, ANOVA and Kruskal-WallisOne Way ANOVA on Ranks. For posthoc pairwise multiple comparisons, eachP value was compared to a critical level depending on the rank of the Pvalue and the total number of comparisons made to determine ifdifferences between groups are significant. Sigma Plot V11.0 (SystatSoftware Inc.) was used for statistical analyses.

The effects of 1D11 on bone and lungs of OI mice were unknown at studystart. To determine the initial sample size per group of mice wecalculated that to detect a minimal difference of 20% in bone mass(BV/TV) by MicroCT between 1D11 and control treated OI mice with a 90%power, a group size of 8 mice is required.

Example 1: Altered TGFβ Signaling in Crtap^(−/−) Calvaria

Crtap^(−/−) mice and age-matched wild type (WT) littermate controls wereanalyzed for expression of activated pSmad 2, a member of the TGFβsignaling pathway, as well as other downstream targets of TGFβ. Calvariabones were excised and RNA and protein were extracted and analyzed byRealtime-PCR and Western blot, respectively. As can be seen in FIGS. 1Aand 1B, Crtap^(−/−) mice had a 100% higher ratio of activated pSmad2 tototal Smad2 compared to WT mice, as measured by Western blot andquantified by densitometry, indicating that TGFβ signaling is elevatedin Crtap^(−/−) mice. Transcriptional targets of TGFβ, such as Col1a1 andp21, were elevated compared to WT controls, as measured by RT-PCR anddemonstrated in FIG. 1C and FIG. 1D, respectively. The pro-fibrotic ECMprotein connective tissue growth factor (CTGF) was measured and found tobe approximately 50% higher in Crtap^(−/−) mice compared to WT controls,as determined by RT-PCR and demonstrated in FIG. 1E. As shown in FIG.1F, RT-PCR analysis revealed that expression of the cyclin-dependentkinase inhibitor p27 was not altered in either the Crtap^(−/−) or the WTmice.

Example 2: Increased TGFb Activity in Crtap^(−/−) Mice In Vivo and inCrtap^(−/−) Osteoblastic Cells

Crtap^(−/−) mice were crossed to TGFβ reporter mice that expressluciferase in response to activation of TGFβ signaling (JacksonLaboratory; B6.Cg-Tg(SBE/TK-luc)7Twc/J). P9 mice were injected with thesubstrate D-Luciferin (150 mg/kg) 10 minutes before imaging (Xenogen;IVIS camera system). As demonstrated in FIG. 2A, Crtap^(−/−) mice hadconsiderably higher luminescence in their tails, long bones, andcalvaria compared to WT controls, indicating increased TGFb activity inCrtap^(−/−) mice. FIG. 2B, quantification of luciferase activity atcalvaria.

Bone marrow stromal cells (BMSCs) were isolated from Crtap^(−/−) miceand WT mice, cultured under osteogenic conditions ex vivo, and theconditioned culture medium was analyzed for TGFβ activity using a cellline that expresses luciferase in response to activation of TGFβsignaling. As shown in FIG. 2C, conditioned medium from Crtap^(−/−)BMSCs resulted in nearly a two-fold greater luciferase activity of thereporter cell line compared to BMSCs from WT mice. Together, these dataindicate that TGFβ secretion and activity is elevated in the bones andosteoblastic cells of Crtap^(−/−) mice.

Example 3: μCT Analysis of Crtap^(−/−) Vertebrae

Adult 8 week old Crtap^(−/−) mice (N=6 per group) were administered 1D11(10 mg/kg, I.P., 3 times/week, 8 weeks total), a murine surrogate of thepan-specific antibody that binds to TGFβ comprising a heavy chaincomprising the amino acid sequence of SEQ ID NO: 14 and a light chaincomprising the amino acid sequence of SEQ ID NO: 15. An unrelated 13C4antibody was administered to a separate group of Crtap^(−/−) mice and WTmice as a control (N=6). The L4 vertebral bodies of 16 week old mice(treated from week 8-16) were imaged by μ-CT. MicroCT data of vertebralbody L4 from 8 week old Crtap^(−/−) mice (n=6 per group) that weretreated with the TGFβ neutralizing antibody 1D11 (Genzyme; 10 mg/kg,I.P., 3 times/week) for 8 weeks and wild-type (WT) and controlCrtap^(−/−) mice that were treated with a control antibody(13C4-placebo) is shown in FIG. 3. As shown in FIG. 3, Crtap^(−/−)vertebrae were cavernous compared to WT control vertebrae. However, 1D11treatment result in a skeletal phenotype that was comparable to the WTcondition.

The data from FIG. 3 was quantified in FIG. 4, treatment with thepan-specific anti-TGFβ antibody rescued the skeletal phenotype ofCrtap^(−/−) mice. Vertebrae from treated Crtap^(−/−) mice werestatistically similar to WT control mice in measured parameters,including bone volume density (BV/TV), total bone surface (BS), bonesurface density (BS/BV), trabecular number (Tb.N), trabecular thickness(Tb.Th), trabecular spacing (Tb.Sp), and total volume (Dens TV).

Example 4: Histomorphometry of Anti-TGFβ Treated Crtap^(−/−) Vertebrae

In addition to μ-CT, the vertebral bones of antibody-treated,placebo-treated, and WT mice were analyzed by histomorphometry. As shownin FIGS. 5A and 5B, μ-CT results were confirmed by histomorphometricanalysis. In addition, tissue sections were stained for expression ofthe osteoclast marker TRAP. Analysis revealed that there were moreosteoclasts covering more of the bone surface in Crtap^(−/−) micecompared to WT controls (N.Oc/BS and Oc.S/BS), indicating increasedosteoclastic activity. Treatment with the 1D11 anti-TGFβ reduced allosteoclast-specific parameters to below WT numbers. Thus, osteoclastswere identified as a potential target for TGFβ antibodies, and morespecifically, pan-specific anti-TGFβ antibodies.

Example 5: Three-Point Bending Test of Anti-TGFβ Treated Crtap^(−/−)Femurs

Biomechanical testing was performed on the excised femurs of 16 week oldmice (after treatment from week 8-16) using a standard three-pointbending test with an Instron 5848 device (Instron Inc., Norwood Mass.)with a 6 mm span, preloaded to 1N at a rate of 1N/s for 5 seconds.Following the pre-loading, femurs were compressed to failure at a rateof 0.1 mm/sec. Load and displacement data were captured at rate of 40 Hzby using BLUEHILL Software (Instron 5848).

As demonstrated in FIG. 6, Crtap^(−/−) mice femurs were less stiff andwere able to withstand a significantly smaller maximal load compared toWT control mice. Femurs of 1D11 treated Crtap^(−/−) mice showed asignificant improvement of the maximum load, and a trend to an increasedstiffness compared to control crtap^(−/−) mice.

Thus, treatment with the 1D11 pan-specific anti-TGFβ antibodyquantitatively, qualitatively, and biomechanically restored the skeletalphenotype of Crtap^(−/−) mice.

Example 6: Inhibition of TGFβ Signaling with 1D11 Ameliorates the LungPhenotype

Crtap^(−/−) mice have a generalized connective tissue disease manifestedby low bone mass, glomerulosclerosis, and pulmonary dysplasia (Baldridgeet al.; PloS One, 5(5):e10560 (2010)). Increased TGFβ expression wasseen the lungs of Crtap^(−/−) mice, as evidenced by positiveimmunostaining for pSmad2 and demonstrated in FIG. 7A. Histologically,Crtap^(−/−) mice exhibited increased distal airway space compared to WTmice, as .shown in FIG. 7B. 1D11 treatment (10 mg/kg, IP, 3×/week for 8weeks) reduced pSmad2 expression in Crtap^(−/−) mice and reduced thedistal airway space and ameliorated the lung phenotype, as demonstratedin FIG. 7A and FIG. 7B and quantified in FIG. 7C (*P<0.05 vs. controlCrtap^(−/−); 10 images analyzed per mouse, n=8 mice per group).

Example 7: Decorin Expression in Crtap^(−/−) Lungs

Transcriptional regulators of TGFβ expression were investigated in orderto understand the basis of dysregulated TGFβ signaling in the bones andlungs of Crtap^(−/−) mice. A major class of extracellular proteins thatcan regulate TGFβ in ECM include the small leucine rich proteoglycans(SLRP), such as decorin. Immunostaining revealed increased expression ofdecorin in Crtap^(−/−) lungs compared to WT control lungs, as shown inFIG. 8.

As decorin is a regulator of mature TGFβ, this finding suggests thataltered post-translational modification of collagen, as occurs in OI,alters the interactions of ECM proteins, including SLRPs. Decorin bindsto hydroxyproline sites, such as the one located at amino acid residue396 of type-I and type-II collagens, which are absent in Crtap^(−/−)mice. A decorin binding assay was performed to determine whether decorinbinding may be altered in OI, and whether this may be at least partlyresponsible for the phenotypes observed in Crtap^(−/−) bones and mice.As shown in FIG. 9, decorin binding to 3-hydroxylated collagen peptides(as in type I and II collagen) was greater than collagen peptideswithout 3-hydroxylation (as in type III collagen).

Example 8: Increased TGFβ Signaling is a Common Mechanism inOsteogenesis Imperfecta

OI is characterized by brittle bones, low bone mass, bone deformitiesand fractures. In addition, extraskeletal manifestations including lungabnormalities contribute substantially to morbidity and mortality. Mostcases of OI are caused by autosomal dominant mutations in the genesencoding type I collagen (COL1A1 and COL1A2). In recent years, mutationsin additional genes encoding the proteins involved in thepost-translational modification of collagen have been identified ascausing recessive forms of OI. The first described was in cartilageassociated protein (CRTAP), a member of the prolyl-3-hydroxylase complexthat is responsible for 3-hydroxylation of proline residue 986 α1(I) intype I collagen. Hypomorphic CRTAP mutations lead to partial loss of3-hydroxyproline (3Hyp) in fibrillar collagen as well asovermodification of other residues and result in recessive OI type VII,which clinically overlaps with dominant forms of severe OI. Thephysiological function of 3Hyp is not completely understood, butbiochemical studies suggest that it may be involved in collagen-proteininteractions, rather than negatively affecting collagen stability.

The ECM is an important reservoir for signaling molecules and theirregulators. In bone, TGFβ acts as a central coordinator of boneremodeling by coupling the localized activity of bone resorbingosteoclasts and bone forming osteoblasts. TGFβ is abundantly produced byosteoblasts, is secreted predominantly in inactive latent forms, and isdeposited into the bone matrix. Here, it can be released and activatedduring bone resorption by osteoclasts. As an additional level ofregulation, active TGFβ can be bound by proteoglycans, which modulateits bioactivity in association with collagen fibrils. Because type Icollagen is the most abundant component of the ECM in bone, this raisesthe intriguing hypothesis that the alteration of collagen structureobserved in OI not only increases bone fragility, but also affects thesignaling reservoir function of the bone matrix. Interestingly,Crtap^(−/−) mice show phenotypic overlap with animal models of increasedTGFβ signaling. For example, TGFβ overexpression results in low bonemass. In addition, Crtap^(−/−) mice exhibit an enlargement in alveolarairway space in lungs, which is similar to that observed in a mousemodel of Marfan syndrome, where increased TGFβ signaling has been shownto be a major contributor to the lung pathology. Therefore, the statusof TGFβ signaling was studied in the Crtap^(−/−) mouse model ifrecessive OI.

To assess the status of TGFβ signaling in bone, the expression levels ofTGFβ target genes in calvarial bone of Crtap^(−/−) mice were evaluated.Compared with wild type (WT) samples, Crtap^(−/−) bone showed anincreased expression of the TGFβ downstream targets p21(cyclin-dependent kinase inhibitor 1), PAI-1 (plasminogen activatorinhibitor-1), and Col1a1, consistent with elevated TGFβ activity (FIG.10A). To confirm increased activation of the intracellular TGFβsignaling pathway, the status of Smad2, an intracellular secondmessenger protein, which becomes phosphorylated after activation of TGFβreceptors, was evaluated. Consistent with target gene expression,immunoblot analyses demonstrated a higher ratio of phosphorylated Smad2(pSmad2) to total Smad2 in bone samples of Crtap^(−/−) mice, indicatingincreased TGFβ signaling (FIGS. 10B and 10C).

To determine whether these static measures reflect increased TGFβactivity in vivo, Crtap^(−/−) mice were intercrossed with TGFβ-reportermice expressing luciferase under control of TGFβ-responsive Smad bindingelements (SBE-Luc mice). Compared with WT/SBE-Luc littermates,Crtap^(−/−)/SBE-Luc mice showed an increase in bioluminescence of areasover skeletal structures, indicating increased TGFβ activity in vivo(FIG. 10D). In 3 litters, Crtap^(−/−) mice show a mean 2.86 fold(SD±0.34) bioluminescence signal at the head/calvaria compared with WTmice. Moreover, in order to test whether the increased TGFβ/Smadsignaling associated with loss of Crtap is intrinsic to bone, i.e.,tissue autonomous, bone marrow stromal cells (BMSCs) were differentiatedto osteoblastic cell in vitro. By using a TGFβ reporter cell line, itwas found that conditioned medium from Crtap^(−/−) BMSCs exhibitedhigher TGFβ activity compared with medium from WT BMSCs (FIG. 10E).Together, these findings indicate that loss of Crtap enhances TGFβsignaling in bone in a tissue autonomous fashion.

Patients with severe OI can also exhibit intrinsic lung abnormalities,and respiratory failure is one of the leading causes of death in theseindividuals. Interestingly, Crtap^(−/−) mice show a diffuse increase inalveolar airway space, a feature associated with increased TGFβsignaling in other developmental models. Accordingly, lungs of Crtapmice showed increased intracellular staining for pSmad2 in alveolarcells, indicating that the increased TGFβ activity is also present inextraskeletal tissues (FIG. 10F).

To understand whether increased TGFβ signaling represents a causalmechanism contributing to the bone and lung phenotypes in Crtap^(−/−)mice, a rescue experiment was performed with a pan-TGFβ neutralizingantibody (1D11). Eight week old Crtap^(−/−) mice were treated with 1D11for 8 weeks; control Crtap^(−/−) and WT mice received a non-specificcontrol antibody (13C4). 1D11 did not significantly change body weightof the treated Crtap^(−/−) mice, indicating that TGFβ inhibition did notaffect the general nutritional status (FIG. 14). In addition, massspectrometric and cross-links analyses showed that 1D11 did notsignificantly change the status of type I collagen P986 3-hydroxylationor collagen crosslinks in Crtap^(−/−) mice, suggesting that dysregulatedTGFβ signaling is a consequence of the altered molecular collagenstructure, and not directly involved in intracellular collagenprocessing or extracellular fibril assembly (FIG. 15). Crtap^(−/−) miceexhibit a reduced bone mass and abnormal trabecular bone parameters(FIGS. 11A and 11B). MicroCT imaging analysis of vertebrae demonstratedthat compared with control Crtap^(−/−) mice, TGFβ inhibitionsignificantly improved trabecular bone parameters, including bonevolume/total volume, trabecular number and trabecular thickness to nearWT levels (FIGS. 11A and 11B, and FIG. 17). Similar beneficial effectswere observed in femoral trabecular bone in Crtap mice, where TGFβinhibition significantly improved trabecular bone parameters (FIG. 18).The effects of TGFβ inhibition on the skeleton with 1D11 have beenreported previously in WT mice and in Es1-1^(−/−) mice11, a model withincreased TGFβ activity due to a defect in normal TGFβ maturation. While1D11 moderately increased trabecular BV/TV by 33% in the spine in WTmice, Es1-1^(−/−) mice exhibited an 106% increase in BV/TV. Thissuggested that targeting TGFβ in a pathophysiological situation where itis increased in the skeleton, could lead to a relatively more pronouncedpositive effect. In the present study, 1D11 increased the trabecularBV/TV at the spine by 235% in Crtap^(−/−) mice, supporting that thedysregulated TGFβ signaling is an important contributor of the low bonemass in Crtap^(−/−) mice. At the femur midshaft, the parameters ofcortical architecture including cortical thickness, diameter,cross-sectional area and cross-sectional moments of inertia inCtrap^(−/−) mice were significantly reduced compared to WT mice.Following 1D11 treatment, these parameters were no longer significantlydifferent from WT mice (FIG. 19). To test if these changes in corticaland trabecular bone translated into improved bone strength,biomechanical testing of the femurs by 3-point bending was performed. Itwas found that TGFβ inhibition was able to increase maximum load andultimate strength in the treated Crtap^(−/−) mice, indicating improvedwhole bone and tissue strength and improved resistance to fracture.However, 1D11 treatment had no effects on the increased brittleness ofthe OI bone, as indicated by the reduced post-yield displacement in bothcontrol and 1D11 treated Crtap^(−/−) mice (FIG. 20). This likelyreflects the inherent abnormal mineralization associated with alteredcollagen structure. Taken together, these findings indicate thatincreased TGFβ signaling is major contributor to the bone phenotype inrecessive OI resulting from Crtap deficiency and that inhibition ofdysregulated TGFβ signaling restores bone mass, microstructuralparameters and improves whole bone strength.

To understand the effects of TGFβ inhibition in Crtap^(−/−) mice at thecellular level, histomorphometric analyses on treated mice wasperformed. In sections of vertebral bodies in this study it was foundincreased osteoclast (Oc) and osteoblast (Ob) numbers per bone surfacein control Crtap^(−/−) compared to WT mice, indicating increased boneremodeling in the spine (FIG. 11C and FIG. 21). Consistently, the serumbone turnover markers osteocalcin (OCN) and C-terminal cross-linkedtelopeptide of bone collagen (CTX) were elevated in 8 week old (OCN andCTX) and 16 week old (CTX only) control Crtap^(−/−) mice (FIG. 19).Similar changes in the cellular composition of bone have been describedin patients with dominant and recessive OI, showing increased Oc and Obnumbers consistent with an increased bone turnover. Interestingly, mousemodels of increased TGFβ signaling also show low bone mass withincreased osteoclastic bone resorption and abnormal bone remodeling.Most reports of the effects of TGFβ on bone cells are consistent with amodel where TGFβ can stimulate the recruitment and initialdifferentiation of Oc and Ob precursors at the site of bone repair,followed by insulin-like growth factor 1 (IGF-1) mediated Obdifferentiation. However, at persistently high doses, TGFβ can inhibitOb differentiation by repressing the differentiation factor RUNX2. Giventhe crucial effects on Oc/Ob interaction, fine tuning of TGFβavailability is a key factor for the local coupling of bone resorptionwith formation during bone remodeling and its imbalance can lead tosignificant bone pathology.

In contrast to the findings in control Crtap^(−/−) mice, bone sectionsof Crtap^(−/−) mice treated with 1D11 revealed reduced Oc and Obnumbers, which were even lower than the values measured in the WT mice,indicating a supraphysiologic suppression of dysregulated boneremodeling as a result of TGFβ inhibition at the dose of 1D11 used inthis experiment (FIG. 11C). Consistent with an earlier report, theobservation of a reduction of Oc's and Ob's below WT levels alsounderscores the physiological requirement of local amounts of TGFβ tonormally coordinate Oc's and Ob's during the bone remodeling process.Our findings are different from previous studies in WT mice, where 1D11treatment reduced Oc numbers but increased Ob numbers. This may reflectdistinct cellular effects of TGFβ inhibition in a pathophysiologicalsituation with increased TGFβ signaling and increased bone remodelingcompared with normal bone in WT mice. TGFβ has been shown to inhibitdifferentiation of osteoblast precursor cells, and increased TGFβsignaling could thereby lead to a higher proportion of immatureosteoblast lineage cells. On the other hand, an increased number orhigher proportion of immature Ob's on the bone surface could result inan increased amount of secreted TGFβ by these cells. The finding thatTGFβ inhibition with 1D11 significantly reduces the increased Ob numbersin Crtap^(−/−) mice suggests that the increased TGFβ signaling causallycontributes to the increase in osteoblast lineage cells.

In addition to the findings regarding Oc and Ob numbers, greaterosteocyte (Ot) numbers per bone area in control Crtap^(−/−) mice wereobserved, which were reduced to levels comparable with those of WT micein 1D11 treated Crtap^(−/−) mice (FIG. 11C and FIG. 21). In OI patients,an increased Ot density has been observed in individuals with moresevere forms of the disease, likely reflecting the presence of immatureprimary bone due to a defect in physiological maturation in OI bone.Consistent with our hypothesis that increased TGFβ signaling contributesto the bone pathology in OI, overexpression of TGFβ in WT mice similarlyresults in an increased Ot density. As a possible explanation, TGFβ caninhibit Ob apoptosis during the transition of Ob's to Ot's, and therebylead to an increased Ot density. Collectively, these findings indicatethat increased TGFβ signaling contributes to a high bone turnover statusand impaired bone maturation in Crtap^(−/−) mice and that inhibition ofdysregulated TGFβ signaling reverses these cellular alterations.

Given these crucial effects on Oc/Ob interaction, fine tuning of TGFβavailability is a key factor for the local coupling of bone resorptionwith bone formation during bone remodeling and its imbalance can lead tosignificant bone pathology. Our findings indicate that inhibition ofdysregulated TGFβ signaling in Crtap^(−/−) mice restores bone mass aswell as microstructural parameters, improves whole bone strength, andreverses the cellular alterations observed in Crtap^(−/−) mice.Therefore, dysregulation of TGFβ signaling is an important contributorto the bone phenotype in this mouse model of recessive OI.

We were also interested in whether TGFβ inhibition affected the lungphenotype of Crtap^(−/−) mice. Lungs of control Crtap^(−/−) mice show anincrease in the distal airway space compared with WT mice (FIG. 11D).Interestingly, lungs of Crtap^(−/−) mice treated with the TGFβneutralizing antibody showed a 60% improvement in the distance betweenalveolar structures (FIGS. 11D and 11E). This finding indicates thatexcessive TGFβ signaling is also an important pathogenic contributor tothe lung abnormalities present in Crtap^(−/−) mice. Increased TGFβsignaling has been linked to developmental pulmonary abnormalities aswell as disease in mature lungs. For example, TGFβ overexpression inlungs results in impaired lung development with areas of enlarged airwayspace and increased TGFβ signaling is a contributing pathomechanism inlung abnormalities in Marfan syndrome as well as in the development ofemphysema and bronchial asthma. Our results indicate that excessive TGFβsignaling is an important pathogenic contributor to the lungabnormalities present in Crtap^(−/−) mice. Given the partial rescue ofthe lung phenotype with 1D11 in Crtap^(−/−) mice, it is possible thatdysregulated TGFβ signaling affects pulmonary tissue development whenthe anatomic structures are established, in addition to maintaining lungtissue at later stages when TGFβ inhibition is able to ameliorate thephenotype.

The next question asked was how alterations in collagen due to loss ofCrtap (leading to the loss of 3Hyp at P986 and post-translational overmodification of collagen) result in dysregulated TGFβ signaling.Biochemical analyses indicate that collagen prolyl-3-hydroxylation doesnot fundamentally affect the stability of collagen molecules, butinstead it may affect collagen-protein interactions. An attractivehypothesis is that loss of 3Hyp could affect collagen interaction withsmall leucine-rich proteoglycans (SLRPs). SLRPs are known to bind toboth type I collagen as well as TGFβ, and thereby modulate TGFβactivity. For example, the SLRP decorin is able to inhibit distincteffects of TGFβ in osteosarcoma cells whereas it enhances TGFβ activityin preosteoblastic cells. The binding region of decorin on type Icollagen is suggested to center at residues 961/962 of the triplehelical domain, which is located in close proximity to the P986 residue,that is unhydroxylated in OI due to Crtap deficiency. Therefore, it ispossible that the P986 3Hyp position marks an interacting site for thebinding of decorin to type I collagen, thereby mediating thesequestration of mature TGFβ to collagen.

Hence, it was hypothesized that decorin binding to collagen is criticalfor TGFβ regulation and that this binding is disrupted with alteredcollagen structure, for example by loss of post-translational3-prolyl-hydroxylation modification of P986 in the α1 chain of type Icollagen in the case of recessive OI. It was identified that althoughloss of Crtap did not alter the RNA expression of decorin and otherSLRPs in calvarial bone (FIG. 12A), nor the qualitative abundance ofdecorin in trabecular bone (FIG. 24), it did reduce binding ofrecombinant decorin core protein to type I collagen isolated fromCrtap^(−/−) mice versus WT mice (FIG. 12B). Surface plasmon resonanceanalysis measurements of the binding of recombinant decorin core proteinto type I collagen of WT and Crtap^(−/−) mice demonstrated reducedbinding in Crtap^(−/−) mice at the three concentrations tested (FIG.23). Three technical replicates at each of the indicated concentrationsof decorin were performed from two independent biological replicates (♦replicate 1, ▴ replicate 2). Results are shown as the percentage of themean of WT (bars indicate mean per group). The mean reductions ofdecorin binding to Crtap^(−/−) type I collagen at 3, 5 and 12 μM ofdecorin were 28.5%, 33.5% and 38.1%, respectively.

This finding suggests that alterations of collagen-proteoglycaninteractions may contribute to the dysregulated TGFβ signaling in boneand other collagen rich tissues in OI. Based on the reported requirementof decorin-collagen binding for decorin to effectively reduce TGFβbioactivity, it is possible that the defects in OI collagen lead toaltered binding of decorin, and hence, its ability to sequester TGFβ inthe matrix and modulate TGFβ functions. Hence, alteredproteoglycan-collagen interactions may contribute to dysregulated TGFβsignaling in bone and other collagen rich tissues in OI, even if nomajor changes in absolute TGFβ levels are present (FIG. 24 and FIG. 25).This notion is supported by the finding that COL1A1 and COL1A2 mutationsin more severe forms of dominant OI cluster in specific regions that areknown to bind proteoglycans, further supporting a physiologicalrelevance of proteoglycan-collagen interactions for normal bonehomeostasis. This would also imply that other proteoglycans that arecompeting with decorin for the collagen binding site may also contributeto dysregulated TGFβ activity, and that additional signaling pathwayscould be altered.

Because of the clinical overlap of some recessive and dominant forms ofOI where defective structure of collagen fibers leads to brittle bonesand extraskeletal manifestations, it is possible that dysregulation ofTGFβ signaling is a common pathophysiological disease mechanism. Toaddress this hypothesis, the status of TGFβ signaling in a mouse modelof dominant OI was investigated. Knock-in mice carrying a G610C mutationin the Colla2 gene (Col1a2^(tm1.1Mcbr)) phenocopy a dominantlyinherited, moderate form of OI that was originally identified in anAmish population. In bone samples of Col1a2^(tm1.1Mcbr) mice, increasedexpression of the TGFβ target genes p21 and PAI-1 was found, indicatingupregulation of TGFβ signaling (FIG. 13A). Consistently, immunoblotanalyses of bone extracts from Col1a2^(tm1.1Mcbr) mice also showed anincreased ratio of activated pSmad2/total Smad2, similar to ourobservation in the Crtap^(−/−) mice (FIGS. 13B and 13C).

To test if the increased TGFβ signaling in this model of dominant OIalso represents a causal mechanism, 8 week old Col1a2^(tm1.1Mcbr) micewere treated with the TGFβ-neutralizing antibody 1D11 for 8 weeks;control Col1a2^(tm1.1Mcbr) and WT mice were treated with the controlantibody 13C4. Similar to the findings in Crtap^(−/−) mice,1D11-treatment restored the trabecular bone parameters at the spine toWT levels (FIGS. 13D, 13E, and 22). Taken together, these findingsindicate that dysregulated TGFβ signaling is also an importantcontributor to the pathogenesis of dominant forms of OI, and thatanti-TGFβ therapy corrects the bone phenotype in dominant OI.

From a clinical-translational perspective, potential negative effects ofsystemic TGFβ inhibition in OI patients have to be considered. WhileTGF-β1^(−/−) mice develop a severe multifocal inflammatory disease anddysregulation of the immune system within the first weeks of life, inboth Crtap^(−/−) and Col1a2^(tm1.1Mcbr) mice treated with 1D11 we didnot observe obvious negative effects on general health, behavior orgrowth, suggesting that the effects of a partial pharmacologicalinhibition of TGFβ ligands in adult mice are different from a completeloss of TGFβ1 during development. In humans, Fresolimumab (GC1008,Genzyme), which is similar to 1D11 in its affinity and specificity tothe 3 isoforms of TGFβ, was used in phase I studies in patients withtreatment-resistant primary focal segmental glomerulosclerosis,idiopathic pulmonary fibrosis and malignant melanoma or renal cellcarcinoma. In these studies, Fresolimumab was in general well-tolerated,with possible, dose-related adverse events including skin rashes orlesions, epistaxis, gingival bleeding and fatigue.

The molecular mechanisms of OI are incompletely understood. As a result,current treatment options for patients with OI are mainly limited toanti-resorptive therapies as used for the treatment of osteoporosis.Interestingly, a recent randomized, placebo controlled trial of theanabolic agent teriparatide in adults with OI showed that severe OI typeIII/IV responded differently than did those with mild OI type I (Orwollet al., 2014). This suggests genotypic differences in response totherapies targeted at modifying cell signaling and that TGFβ-targetedtreatment may be a promising option to further study in severe OI due tocollagen and collagen post-translational modification gene mutations.Overall, our data support the concept of dysregulated matrix-cellsignaling as a mechanism in the pathogenesis of different geneticallyinherited forms of brittle bone disease and point to a disease-specificmechanism-based strategy for the treatment of OI by neutralizing theoveractive TGFβ activity in skeletal and extraskeletal tissues.

1-24. (canceled)
 25. A method of treating osteogenesis imperfecta (OI)in a human subject in need thereof, comprising: administering to thesubject an antibody or an antigen-binding fragment thereof that binds totransforming growth factor β (TGFβ), wherein the antibody orantigen-binding fragment comprises heavy chaincomplementarity-determining regions (CDRs) 1-3 having complementaritydetermining regions (CDRs) 1-3 having the amino acid sequences of SEQ IDNOs: 4, 5, and 6, respectively; and light chain CDR1-3 having the aminoacid sequences of SEQ ID NOs: 7, 8, and 9, respectively; and measuringthe level of a bone parameter selected from the group consisting of bonevolume density (BV/TV), total bone surface (BS), bone surface density(BS/BV), trabecular number (Tb.N), trabecular thickness (Tb.Th),trabecular spacing (Tb.Sp), and total volume (Dens TV).
 26. The methodof claim 25, wherein the antibody comprises a heavy chain variableregion (V_(H)) and a light chain variable region (V_(L)) comprising theamino acid sequences of SEQ ID NOs: 10 and 11, respectively.
 27. Themethod of claim 25, wherein the antibody comprises a human IgG₄ constantregion and a human κ light chain constant region.
 28. The method ofclaim 25, wherein the antibody comprises a heavy chain and a light chaincomprising the amino acid sequences of SEQ ID NOs: 14 and 15,respectively.
 29. The method of claim 25, wherein the antibody orantigen-binding fragment thereof inhibits bone resorption.
 30. Themethod of claim 25, wherein the antibody or antigen-binding fragmentthereof promotes bone deposition.
 31. The method of claim 25, whereinthe antibody or antigen-binding fragment thereof improves the functionof a non-skeletal organ affected by OI selected from the groupconsisting of hearing function, lung function, and kidney function. 32.The method of claim 25, wherein the subject has type I OI.